Arabidopsis FHY3 and FAR1 integrate light and strigolactone signaling to regulate branching

Xie, Yurong; Liu, Yang; Ma, Mengdi; Zhou, Qin; Zhao, Yongping; Zhao, Binbin; Wang, Baobao; Wei, Hongbin; Wang, Haiyang

doi:10.1038/s41467-020-15893-7

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Published: 23 April 2020

Arabidopsis FHY3 and FAR1 integrate light and strigolactone signaling to regulate branching

Yurong Xie ORCID: orcid.org/0000-0001-6476-2467¹,
Yang Liu¹,
Mengdi Ma¹,
Qin Zhou²,
Yongping Zhao²,
Binbin Zhao²,
Baobao Wang¹,
Hongbin Wei³ &
…
Haiyang Wang ORCID: orcid.org/0000-0002-1302-5747^3,4

Nature Communications volume 11, Article number: 1955 (2020) Cite this article

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Abstract

Branching/tillering is an important parameter of plant architecture and is tightly regulated by both internal factors (such as plant hormones) and external factors (such as light conditions). How the various signaling pathways converge to coordinately regulate branching is not well understood. Here, we report that in Arabidopsis, FHY3 and FAR1, two homologous transcription factors essential for phytochrome A-mediated light signaling, and SMXL6/SMXL7/SMXL8, three key repressors of the strigolactone (SL) signaling pathway, directly interact with SPL9 and SPL15 and suppress their transcriptional activation of BRC1, a key repressor of branching, thus promoting branching. In addition, FHY3 and FAR1 also directly up-regulate the expression of SMXL6 and SMXL7 to promote branching. Simulated shade treatment reduces the accumulation of FHY3 protein, leading to increased expression of BRC1 and reduced branching. Our results establish an integrated model of light and SL coordinately regulating BRC1 expression and branching through converging at the BRC1 promoter.

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Introduction

Branching (tillering in cereal crops) is a major component of plant architecture and critical determinant of crop productivity. A critical stage of branch development is bud outgrowth after the formation of axillary buds, and this process is intricately regulated by endogenous developmental cues and various environmental signals¹. Recent molecular genetic studies have revealed a conserved TB1/FC1/BRC1 pathway repressing axillary bud outgrowth in both monocots and dicots. TEOSINTE BRANCHED 1 (TB1), which encodes a TCP-family transcription factor, was initially identified as a key repressor of lateral branching in maize². Later studies showed that loss-of-function mutants of the genes homologous to TB1 in rice and Arabidopsis (FINE CULM1 or FC1 in rice, Branched1 or BRC1 in Arabidopsis) display a similar excess tillering/branching phenotype^3,4,5. Moreover, recent studies showed that strigolactone (SL), a newly identified plant hormone, represses bud outgrowth in both monocots and dicots^6,7,8. Further, it was shown that SLs repress tiller outgrowth in rice through promoting the degradation of a central repressor protein, D53^9,10. Similarly, it has also been shown that in Arabidopsis, SL-induced degradation of three D53-like SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKE proteins SMXLs (SMXL6, SMXL7, and SMXL8) leads to activation of BRC1 to inhibit branching¹¹.

Substantial evidence suggests that the MIR156-SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) module plays an important role in regulating diverse aspects of plant growth and development, ranging from vegetative to reproductive phase transition, branching, leaf development, flowering time, panicle/tassel architecture, fruit ripening, fertility, lateral root development, and abiotic stress responses^12,13. SPLs encode a family of plant-specific transcription factors that share a highly conserved DNA-binding domain, the SBP domain. It has been shown that Arabidopsis SPL9 and SPL15 negatively regulate branching by suppressing axillary meristem initiation and that their loss-of-function mutants display an enhanced branching phenotype^14,15. Similarly, a single-nucleotide polymorphism that escapes miR156 targeting or increase OsSPL14 expression via epigenetic regulation confer an ideal plant architecture to rice, including fewer unproductive tillers, stronger culm, enlarged panicle and ultimately, enhanced grain yield. Thus OsSPL14 was also named Ideal Plant Architecture1 (IPA1) or WEALTHY FARMER’ S PANICLE (WFP)^16,17 (IPA1 was used hereafter). Further analysis revealed that IPA1 could directly bind to the promoter of OsTB1 (FC1), to regulate branching/tillering¹⁸. Recent studies further showed that IPA1 can directly bind to the promoter of D53 and that D53 protein can inhibit the transcriptional activation activity of IPA1 by direct physical interaction, thus forming a feedback loop to repress the expression of D53¹⁹. Such a mechanism has also been shown to be conserved in wheat²⁰. These results clearly indicate that the SL signaling pathway and the MIR156/SPL regulatory module converge at the promoter of TB1/FC1/BRC1 to coordinately regulate branching in plants.

Light is a major environmental factor that regulates branching pattern in plants. When plants sense a reduction in red to far-red light ratio (R/FR) due to competition for light from the neighbor plants (vegetation proximity) or grown under canopy shade, they initiate a set of adaptive responses collectively termed shade avoidance syndrome (SAS), including rapid shoot elongation, more erect leaves, accelerated leaf senescence, early flowering, reduced branching, and ultimately decreased biomass and seed yield if the shading is persistent^21,22. It has been shown that under simulated shade conditions (low R/FR ratios), the expression of BRC1 is elevated, leading to suppression of branching activity²³. We previously showed that under simulated shade conditions, accumulation of the phytochrome-interacting factors (PIFs) proteins increases, and they directly bind to the promoters of several MIR156 genes and repress their expression, thus releasing the downstream SPL transcription factors to regulate a wide range of physiological responses, including branching²⁴. In addition, earlier studies reported that mutations in Arabidopsis FHY3 and FAR1, which encode two homologous transcription factors essential for phytochrome A (phyA)-mediated far-red light signaling in Arabidopsis^25,26,27, caused reduced branching number²⁸. Despite the progress made in this area, however, how the light signaling pathway integrates with the SL and MIR156/SPL regulatory module to coordinately regulate branching remains poorly understood.

In this study, we report that FHY3 and FAR1 directly interact with SPL9 and SPL15 and inhibit the binding of SPL9 and SPL15 to the BRC1 promoter, whereas the D53-like proteins SMXL6/7/8 directly interact with SPL9 and SPL15 and suppress their transactivation activity on BRC1, thus promoting lateral bud outgrowth and branching. In response to simulated shade conditions, FHY3 and FAR1 protein levels reduce, which on one hand leads to the release of SPL9 and SPL15 to bind to BRC1 promoter and activate its expression, and on the other hand, leads to reduced expression of SMXL6 and SMXL7, which is directly upregulated by FHY3 and FAR1, resulting in elevated expression of BRC1 and suppression of branching. Our results establish a complex regulatory network regulating branching under shade conditions through integrating the light and SL signaling pathways with the MIR156/SPL regulatory module.

Results

FHY3 and FAR1 regulate branching in response to shade

Previous studies reported that fhy3 loss-of-function has reduced lateral branching²⁸, indicating that FHY3 is a positive regulator of branching. To confirm this, we firstly compared the number of rosette branches (longer than 2 mm) in wild type (WT), fhy3-11 and far1-4 single mutants, the fhy3 far1 double mutant, and the FHY3 overexpressor (FHY3-OE) under normal high R/FR (white light, WL) conditions. Our observation showed that the FHY3-OE plants had much more rosette branches (27.3% more than that of WT), while the mutant plants all had less branches compared with the WT (only 16.4% and 13.4% of WT for fhy3-11 and fhy3 far1, respectively) (Fig. 1a, b). Further RT-qPCR analysis showed that the transcript level of BRC1 was significantly higher in both the fhy3-11 and fhy3 far1 plants but obviously lower in the FHY3-OE plants compared with the WT plants (Supplementary Fig. 1).

**Fig. 1: FHY3 and FAR1 play a role in regulating branching in response to light quality change.**

To test whether FHY3 and FAR1 play a role in shade induced repression of branching, we compared the number of rosette branches of WT, fhy3 far1 double mutant and FHY3-OE plants. We treated the plants with far-red light (15 μmol m⁻² s⁻¹) for 30 min at the end of each light period before returning to darkness (EOD-FR) to simulate the shade conditions²⁹. Seven-day-old seedlings of WT, fhy3 far1 double mutant and FHY3-OE were treated with EOD-FR for 30 min every day before returning to darkness. After 4 week’s treatment, the number of rosette branches (>2 mm in length) was counted. The results showed that compared with their counterparts grown under normal WL conditions, all EOD-FR treated plants had significantly reduced rosette branches (Fig. 1a, b), especially for the WT and FHY3-OE plants (~51% and ~62% reduction for WT and FHY3-OE, respectively), while the fhy3-11 and fhy3 far1 mutants had less decline (~31% and ~27% of reduction for fhy3-11 and fhy3 far1, respectively) (Fig. 1a, b). These observations suggest that the FHY3-OE plants are more sensitive, while the fhy3-11 and fhy3 far1 mutants are less sensitive to the simulated shade treatment than the WT plants. Consistent with this, RT-qPCR analysis revealed that the transcript level of BRC1 was significantly upregulated in all shade-treated materials compared with their counterparts grown under normal WL conditions, with the highest relative increase of expression being detected in the FHY3-OE plants subjected to simulated shade treatment (Supplementary Fig. 1).

We next examined whether the transcript levels of FHY3 and FAR1 are regulated by simulated shade treatment. RT-qPCR analysis revealed that, compared with the control plants, the expression of both FHY3 and FAR1 were significantly downregulated in plants treated with EOD-FR, with ~38.5% and 35.4% reduction for FHY3 and FAR1, respectively (Fig. 1c). To assess the effect of simulated shade treatment on the protein accumulation of FHY3, 7-day-old 35S::FLAG-FHY3-HA transgenic seedlings³⁰ were treated with 15 μmol m⁻² s⁻¹ FR light for 30 min at the end of the light period. The seedlings were harvested at 1, 4, and 6 h post treatment for western blotting analysis. The results showed that accumulation of FHY3 protein rapidly decreased after the EOD-FR treatment (Fig. 1d). Consistent with this, the accumulation of the YFP-FHY3 fusion protein in the pFHY3::YFP-FHY3 transgenic seedlings³¹ also declined in the EOD-FR treated samples (Fig. 1e). We further used proteasome inhibitor MG132 to test whether the decline of FHY3 protein accumulation under simulated shade is regulated by 26S proteasome. Treatment with MG132 significantly slowed down the degradation of FHY3 in the samples treated with simulated shade (Supplementary Fig. 2a, b), indicating that FHY3 protein stability is regulated by the 26S proteasome pathway.

SPL9/15 inhibit branching by repressing BRC1 expression

Previous studies have shown that Arabidopsis SPL9 and SPL15 negatively regulate branching and that their loss-of-function mutants display an enhanced branching phenotype^14,15. To confirm their roles in repressing branching, we generated spl9 spl15 and brc1 brc2 double knockout mutants, transgenic overexpressors of BRC1 (BRC1-OE) and rSPL9 (rSPL9-OE, which expresses a miR156-resistant form of SPL9) (Supplementary Fig. 3). As expected, the spl9 spl15 double mutant and the brc1 brc2 double mutant had more rosette branches than the WT (~39% and ~74.4% more than WT for spl9 spl15 and brc1 brc2, respectively), whereas the rSPL9-OE and BRC1-OE plants had less rosette branches than the WT (~72.4% and ~44.2% of WT for rSPL9-OE and BRC1-OE, respectively) (Fig. 2a and Supplementary Fig. 4). RT-qPCR assay showed that spl9 spl15 had significantly lower BRC1 expression level than WT, while rSPL9-OE had significantly higher BRC1 expression level than WT (Supplementary Fig. 5). To test whether SPL9 and SPL15 repress branching through BRC1 and BRC2, we generated the rSPL9-OE/brc1 brc2 and BRC1-OE/spl9 spl15 high-order mutants via genetic crosses. Phenotypic analysis showed that rSPL9-OE/brc1 brc2 had similar number of branches as the brc1 brc2 double mutant, while BRC1-OE/spl9 spl15 had similar branch number as the BRC1-OE plants (Fig. 2a and Supplementary Fig. 4). RT-qPCR assay showed that brc1 brc2/rSPL9-OE had similar BRC1 expression level as brc1 brc2, whereas BRC1-OE/spl9 spl15 had similar BRC1 expression level as BRC1-OE (Supplementary Fig. 5). These observations support the notion that SPL9 and SPL15 repress branching through regulating the expression of BRC1.

**Fig. 2: SPL9 and SPL15 bind to the *BRC1* promoter and inhibit shoot branching by upregulating *BRC1* expression.**

As previous studies have shown that rice IPA1 can directly bind to the promoter of FC1 and activate its expression, and that bioinformatics analysis revealed the presence of GTAC motifs for SPL protein binding in the BRC1 and BRC2 promoters (Supplementary Fig. 6), we speculated that Arabidopsis SPL9 and SPL15, which are most closely related to IPA1 based on a phylogenetic analysis¹³, may also directly bind to the BRC1 and BRC2 promoters and regulate their expression. Yeast one-hybrid assay showed that both SPL9 and SPL15 could bind to the S1 fragment (which contains two GTAC motifs) of the BRC1 promoter, but not the BRC2 promoter. Mutation of the GTAC core motifs in the S1 fragment of the BRC1 promoter into TTTT abolished the binding, suggesting that the binding is specific (Fig. 2b and Supplementary Fig. 6). To confirm the binding, we performed electrophoresis mobility shift assay (EMSA) using purified recombinant proteins of the SBP domain of SPL9 and SPL15 fused with the glutathione S-transferase (GST) tag (Supplementary Fig. 7), and the results showed that both SPL9 and SPL15 could bind to the biotin-labeled probe of BRC1 (Fig. 2c). Further, we performed chromatin immunoprecipitation (ChIP)-qPCR assay using transgenic plants that constitutively express miR156-resistant forms of SPL9 and SPL15 fused with HA tag at their C terminal (35S::rSPL9-HA and 35S::rSPL15-HA, respectively). As shown in Fig. 2d, the S1 fragment of the BRC1 promoter was significantly enriched in the ChIP samples. Together, these observations indicate that SPL9 and SPL15 can directly bind to the BRC1 promoter.

Next, we carried out a transient expression assay in N. benthamiana leaves to examine the effect of SPL9 and SPL15 on BRC1 transcription. The results showed that the pBRC1::LUC reporter activity was significantly elevated when co-transfected with either the 35S::rSPL9 or the 35S::rSPL15 construct, compared with the control vector (pSPYCE) (Fig. 2e). To verify the physiological relevance of SPL9/15 activation of BRC1 expression in planta, we generated transgenic plants expressing BRC1 driven by its endogenous WT promoter (pBRC1wt::gBRC1) or BRC1 promoter mutated in the SBP-binding site (pBRC1m::gBRC1) in the spl9 spl15/brc1 brc2 and rSPL9-OE/brc1 brc2 backgrounds. We found that the branch number in the brc1 brc2 plants harboring the pBRC1wt::gBRC1 transgene was restored to WT level, whereas the brc1 brc2 plants harboring the pBRC1m::gBRC1 transgene had significantly more branches than the WT plants (similar to brc1 brc2). We also found that the spl9 spl15/brc1 brc2 plants carrying the pBRC1wt::gBRC1 or pBRC1m::gBRC1 transgenes had similar branch number as the spl9 spl15/brc1 brc2 plants (Supplementary Fig. 8). Moreover, we found that rSPL9-OE/brc1 brc2 plants harboring pBRC1wt::gBRC1 transgene had significantly fewer branches than brc1 brc2 and WT plants. However, no significant difference was detected between rSPL9-OE/brc1 brc2 plants harboring the pBRC1m::gBRC1 transgene and the brc1 brc2 mutant plants (Supplementary Fig. 8). Collectively, these results strongly support the notion that SPL9 and SPL15 act to regulate branch number through regulating BRC1 expression via binding to the SBP core motif in the BRC1 promoter.

FHY3 and FAR1 directly interact with SPL9 and SPL15

As FHY3 and FAR1 play an antagonistic role with SPL9 and SPL15 in regulating branching, we wondered whether these proteins physically interact with each other to coordinately regulate BRC1 expression. Yeast two-hybrid assay showed that both FHY3 and FAR1 interacted with both SPL9 and SPL15 (Fig. 3a). Domain deletion analysis revealed that both the FHY3-I (N-terminal domain containing the DNA-binding zinc finger motif) and FHY3-II domains, but not the FHY3-III domain, could interact with SPL9 and SPL15 (Fig. 3a). The interaction between FHY3 and FAR1 with SPL9 and SPL15 was further confirmed using bimolecular fluorescence complementation (BiFC) assay and luciferase complementation imaging (LCI) assay in N. benthamiana leaf epidermis, in vitro pull-down assay and in vivo co-immunoprecipitation (Co-IP) assay (Fig. 3b–d and Supplementary Fig. 9). Domain mapping analysis also revealed that the SBP-box domain of SPL9 and SPL15 is responsible for interacting with FHY3 and FAR1 (Supplementary Fig. 10).

FHY3 and FAR1 inhibit SPL9/15 binding to the BRC1 promoter

We next tested how the interaction between FHY3 and FAR1 with SPL9 and SPL15 may affect the transcriptional regulation of BRC1. Yeast one-hybrid assay showed that inclusion of an FHY3-expressing construct in the yeast obviously inhibited the activation of SPL9 and SPL15 on the BRC1 promoter-driven reporter gene (Fig. 4a), suggesting that the interaction between FHY3 and SPL9/15 may inhibit the binding of SPL9 and SPL15 to the BRC1 promoter. To confirm this, we carried out EMSA by adding the FHY3 protein (FHY3-I domain) to the mixture of SPL-SBP and biotin-labeled BRC1 probe. The results showed that FHY3 protein itself could not bind to the BRC1 probe, but addition of FHY3 protein (but not the MBP control) significantly reduced the amounts of shifted probes caused by binding of SPL9 and SPL15 (Fig. 4b). To further verify the inhibitory role of FHY3 on the DNA-binding activity of SPL9 and SPL15 in vivo, we performed ChIP-qPCR assay using WT, FHY3-OE and fhy3 far1 double mutants carrying the same 35S:rSPL9-HA transgene grown under normal white light conditions or subjected to EOD-FR treatment. Compared with the WT background, the enrichment of BRC1 DNA fragment declined drastically in the FHY3-OE background, but increased in the fhy3 far1 double mutant background (Fig. 4c). In addition, the enrichment of BRC1 DNA fragment was significantly higher in the EOD-FR treated WT and FHY3-OE plants compared with their counterparts grown under normal white light conditions (Fig. 4c and Supplementary Fig. 11). Together, these results strongly support the notion that FHY3 can inhibit the DNA-binding activity of both SPL9 and SPL15 to the BRC1 promoter and that SPL9 (and probably SPL15 as well) has stronger binding activity to the BRC1 promoter under simulated shade conditions.

**Fig. 4: FHY3 inhibits the binding and transactivation function of SPL9 and SPL15.**

Next, we performed a transient expression assay to test the effect of FHY3 on the transcriptional activation activity of SPL9 and SPL15 on BRC1 by co-infiltrating the 35S::rSPL construct and pBRC1::LUC reporter with or without 35S::FHY3 into N. benthamiana leaves. As shown in Fig. 4d, expression of the reporter gene declined significantly in the samples co-injected with 35S::FHY3, suggesting that FHY3 indeed represses the transcriptional activation activity of SPL9 and SPL15 on BRC1.

To provide genetic evidence supporting the above notion, we generated fhy3 far1/spl9 spl15 and FHY3-OE/spl9 spl15 mutant combinations via genetic crosses and compared their rosette branch numbers. The results showed that both fhy3 far1/spl9 spl15 and FHY3-OE/spl9 spl15 had similar number of rosette branches as the spl9 spl15 double mutant (Fig. 4e, f). RT-qPCR assay showed that fhy3 far1/spl9 spl15 and FHY3-OE/spl9 spl15 had similar BRC1 expression levels as spl9 spl15 (Supplementary Fig. 12). These results together support the notion that FHY3 and FAR1 indeed act upstream of SPL9 and SPL15 to promote rosette branching.

SMXL6/7/8 interact with SPL9/15

Previous studies also showed that in rice, D53 (homolog of SMXL6/7/8) can physically interact with IPA1 (homolog of SPL9 and SPL15) and suppress the transcriptional activity of IPA1¹⁹. We thus tested whether SMXL6/7/8 can interact with SPL9 and SPL15. As expected, our yeast two-hybrid assay showed that all these three SMXL proteins could interact with both SPL9 and SPL15, with stronger interaction being observed for SPL9 (Fig. 5a). The interaction between SMXL6/7/8 and SPL9/15 was further verified by LCI assay and BiFC assay (Fig. 5b and Supplementary Fig. 13). We further selected SMXL6 as a test case and confirmed its interaction with SPL9 and SPL15 using in vitro pull-down assay and in vivo Co-IP assay (Fig. 5c, d).

**Fig. 5: SMXL6, SMXL7, and SMXL8 directly interact with SPL9 and SPL15.**

We next investigated whether SMXL proteins could affect the DNA-binding activity of SPL9 and SPL15 to the BRC1 promoter. Both yeast one-hybrid assay and EMSA assay showed that no obvious effect was observed when SMXL6 was added into these assays (Fig. 5e and Supplementary Fig. 14), suggesting that SMXL proteins are unlikely to affect the DNA-binding activity of SPL9 and SPL15. Next, we performed transient expression assay in N. benthamiana leaves to test the effect of SMXL proteins on transcriptional activation of BRC1 expression. The results showed that addition of SMXL6 protein significantly reduced the activation activity of SPL9 and SPL15 on the reporter gene driven by the BRC1 promoter, compared with the addition of the pSPYNE empty vector. Similar results were also observed for the SMXL7 and SMXL8 proteins (Fig. 5f). These results demonstrate that SMXL6/7/8 inhibit the transcriptional activation activities of both SPL9 and SPL15 on BRC1.

To provide genetic evidence for the above notion, we generated smxl6/7/8 triple mutant and SMXL6D (a mutant form of SMXL6 resistant to degradation) overexpressors (SMXL6D-OE) as described¹¹ (Supplementary Fig. 15). As expected, the smxl6/7/8 triple mutant had fewer, while the SMXL6D overexpressors had more branches than the WT (Fig. 6a, b). We next generated smxl6/7/8/spl9/15 quintuple mutant and SMXL6-OE/rSPL9-OE combinations. Phenotypic assay showed that the smxl6/7/8/spl9/15 quintuple mutant had similar number of rosette branches as spl9 spl15, while SMXL6D-OE/ rSPL9-OE had slightly more (~15.7%) rosette branches than rSPL9-OE (Fig. 6a, b). RT-qPCR assay showed that smxl6/7/8/spl9/15 had similar BRC1 transcript level as spl9/15, while SMXL6D-OE/rSPL9-OE had similar BRC1 transcript level as rSPL9-OE (Supplementary Fig. 16). Together, these results suggest that SMXLs act to promote rosette branching through suppressing the transcriptional activation activities of both SPL9 and SPL15 on BRC1.

**Fig. 6: FHY3 and FAR1 directly upregulate the expression of *SMXL6* and *SMXL7*.**

FHY3 and FAR1 directly upregulate the expression of SMXL6/7

As we observed direct interaction between SPL9 and SPL15 with FHY3/FAR1 and SMXL proteins, we wondered whether FHY3 and FAR1 may interact with SMXL proteins as well. Our yeast two-hybrid assay showed that no direct interaction could be detected between FHY3/FAR1 and SMXL6/7/8 (Fig. 5a). Notably, bioinformatics analysis identified at least two putative FHY3/FAR1-binding sites (FBS, CACGCGC) in the first exon of both SMXL6 and SMLX7 (Supplementary Fig. 17). Thus, we tested whether SMXL6 and SMXL7 are direct target genes of FHY3. Yeast one-hybrid assay showed that FHY3 could directly bind to the first exon of SMXL6 and SMXL7 (Fig. 6c). Moreover, transient expression assay in N. benthamiana leaves showed that co-expression of FHY3 significantly enhanced the transcription of LUC reporter genes driven by the 35S minimal promoter fused with the 5′ UTR together with the first exon of SMXL6 and SMXL7 (Fig. 6d and Supplementary Fig. 18). Further, RT-qPCR assay showed that the transcript levels of SMXL6/7 were all downregulated in the fhy3 far1 double mutant, but upregulated in the FHY3-OE plants (Fig. 6e). In addition, the expression levels of SMXL6 and SMXL7 significantly declined in the WT and FHY3-OE plants grown under simulated shade conditions, compared with their counterparts under normal white light conditions (Fig. 6e). These results suggest that FHY3 (and possibly FAR1) can directly upregulate the expression of SMXL6 and SMXL7, and thus likely play a role in SL signaling.

To test whether FHY3/FAR1 and SMXL6/7 could directly regulate BRC1 expression, we first examined whether FHY3/FAR1 and SMXL6/7 can directly bind to the BRC1 promoter. Yeast one-hybrid assay revealed that there was no direct binding of FHY3/FAR1 or SMXL6/7 to the BRC1 promoter (Supplementary Fig. 19). Secondly, we tested whether FHY3/FAR1 and SMXL6/7 can regulate BRC1 activity through protein–protein interaction. Our yeast two-hybrid assay showed that no direct interaction was detected between BRC1 and FHY3/FAR1 or SMXL6/7 proteins (Supplementary Fig. 20). These results suggest that it is unlikely FHY3/FAR1 and SMXL6/7 directly regulate BRC1 expression or BRC1 activity.

Discussion

Previous studies have shown that light and SLs co-regulate many developmental and physiological processes in plants. For example, it has been shown that SL-mediated inhibition of hypocotyl elongation is dependent on phytochrome and cryptochrome signaling pathways^32,33. In addition, earlier studies showed that MAX2, which encodes a subunit of an SCF E3 ligase, acts as a key positive regulator of the SL signaling pathway and inhibits shoot branching, also functions positively in regulating seedling photomorphogenesis and seed germination^34,35. Moreover, it has been reported that high R/FR conditions promote the biosynthesis of SLs, while low R/FR conditions inhibit the biosynthesis of SLs in tomato and Lotus japonicus^36,37. These results collectively suggest that light and SLs coordinately regulate multiple developmental process in an interdependent manner.

In this study, we found that FHY3 and FAR1, two crucial components of phytochrome A (phyA)-mediated far-red light signaling in Arabidopsis, could directly interact with SPL9 and SPL15 in vivo and in vitro, and inhibit their binding to the promoter of the branching integrator BRC1. We also found that SMXL6/7/8, three components of the SL signaling pathway, directly interact with SPL9 and SPL15 and suppress their transactivation activities on BRC1. Moreover, FHY3 and FAR1 can directly upregulate the expression of SMXL6 and SMXL7. Under stimulated shade conditions, the protein levels of FHY3 and FAR1 decline and the transcript levels of both SMXL6 and SMXL7 are downregulated. Based on these results, we propose a putative model in which accumulation of FHY3 and FAR1 proteins decline under stimulated shade conditions, which in turn releases SPL9 and SPL15 proteins from the inhibitory interactions with FHY3/FAR1 and SMXL6/7/8, leading to increased expression of BRC1 and thus reduced branching (Fig. 6f). Our results suggest that Arabidopsis FHY3 and FAR1 act at the nexus of light and SL signaling integration to coordinately regulate branching, thus greatly deepening our understanding of the signaling cross talk mechanisms of light and SL in regulating plant branching and expanding the functional repertoire of FHY3 and FAR1 in regulating plant growth and development.

It is worth mentioning that our previous studies have shown that in response to phytochrome B inactivation under shade conditions, the protein levels of PIFs rapidly increase, and they can directly bind to the promoters of multiple MIR156 genes and repress their expression, thus releasing the downstream SPLs target genes to regulate various aspects of shade avoidance responses, including reduced branching²⁴. In this study, we showed that FHY3 and FAR1 interact with both SPL9 and SPL15 and inhibit their binding to the BRC1 promoter. These results suggest that multiple signaling pathways could simultaneously operate downstream of phytochromes to regulate shade avoidance response. It will be interesting to investigate whether and how the FHY3/FAR1-medited pathway intersects with the PIFs mediated pathway to generate a coherent response in future studies. In this regard, it is interesting to note that bioinformatics analysis revealed that in Arabidopsis, the promoters of MIR156B, SPL3, SPL7, SPL9, and SPL10 contain at least one FHY3/FAR1-binding site (FBS, CACGCGC), hinting a probability that FHY3 and FAR1 may also participate in shade avoidance response through transcriptional regulation of those factors. Besides, the interaction between DELLA proteins (key repressors of the GA signaling pathway) with SPL9 to coordinately regulate floral transition was recently reported³⁸. The possibility of FHY3 and FAR1 in mediating cross talk between light and GA signaling pathway will also be an interesting avenue for future research. Moreover, earlier studies reported that FHY3 promotes branching through an AXR1-dependent pathway²⁸. However, the detailed mechanism still remains unknown and will be worthy of further investigation.

The continuous increase in world population and reduced arable land, accompanied by continuously deteriorated climate conditions poses a significant challenge to secure global food supply in the coming decades³⁹. Increasing planting density has been an effective means of increasing crop yield per unit land area^40,41. However, shade avoidance response, which is triggered in response to vegetative proximity or canopy shade, is detrimental to crop productivity. Emerging evidence supports the notion that phytochrome signaling pathway has been target of selection during crop domestication and genetic improvement⁴². In addition, it has been shown that in response to shade, inactivation of phytochrome B leads to increased TB1 expression and suppression of axillary bud outgrowth in sorghum^43,44 and that maize grassy tillers1 acts downstream of phyB to suppress axillary bud outgrowth in response to shade signals⁴⁵. Further, the SPL gene family has been proposed to be promising targets of genetic manipulation to improve plant architecture and other agronomic traits tailored for high-density planting^12,13. Given the conserved TB1/FC1/BRC1 pathway in regulating branching/tillering in monocots and dicots, the results described in this report should have important implications for future design of crops with improved plant architecture and thus increased yields, particularly under high-density planting conditions.

Methods

Plant material and growth conditions

The Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used in this study. The mutants spl9-4 (SAIL_150_B05), spl15-1 (SALK_074426), fhy3-11 (SALK_002711), far1-4 (SALK_031652), brc1-2 (SALK_091920), and brc2-1 (SALK_023116) were obtained from the Nottingham Arabidopsis Stock Centre. The T-DNA insertion sites of these mutant alleles were shown in Supplementary Fig. 21. After surface sterilization, seeds were sown on half strength Murashige and Skoog media plates supplemented with 1% sucrose. After a 3-day stratification period in the dark at 4 °C, the plates were then placed in a growth chamber (Percival, USA) at 23 °C with a 16-h-light/8-h-dark photoperiod to allow seed germination and seedling growth.

N. benthamiana seeds were directly sown into soil, germinated in darkness for 4 days, and grown in a culture room at 25 °C with a 16-h-light/8-h-dark photoperiod (light intensity of 100 μmol m⁻² s⁻¹).

EOD-FR treatment

For simulated shade treatment, seedlings were treated with 15 μmol m⁻² s⁻¹ FR light at the end of the light period (EOD-FR) for 30 min every day until harvesting or phenotypic examination.

Rosette branches measurement

For phenotypic investigation, axillary bud and branches were determined as described in Aguilar-Martinez et al.⁵ and Stirnberg et al.²⁸. At least 12 individuals for each genotype were grown under normal conditions or simulated shade conditions for 4 weeks after germination and then the number of rosette branches (longer than 2 mm) was counted.

Plasmid construction

For yeast one-hybrid assay, the full-length coding sequence of SPL9 or SPL15 was PCR amplified from WT cDNA and ligated to the pJG4-5 vector (Clontech, USA) to produce the AD-SPL9 and AD-SPL15 constructs. Promoter fragments (~3 kb in length) of BRC1 or BRC2 were amplified from WT genomic DNA and ligated to the pLacZi2μ vector²⁷ to generate the 2 μ-pBRC1 or 2 μ-pBRC2 constructs. For yeast two-hybrid assay, the coding regions of FHY3, FAR1, or SMXLs were amplified and cloned into the pEG202 vector (Clontech, USA) at the EcoRI restriction site to generate the corresponding LexA fusion construct.

For BiFC, the coding sequences of SPL9 or SPL15 (without stop codon) were cloned into the pSPYCE vector⁴⁶ to generate the 35S::SPL-SPYCE constructs. The coding regions of FHY3, FAR1, and SMXLs (without stop codon) were cloned into the pSPYNE vector to produce the corresponding SPYNE fusion constructs. For LCI assay, the coding sequences of SPL9 or SPL15 were cloned into the pCAMBIA1300-cLUC vector and the coding sequences of FHY3, FAR1, or SMXLs were cloned into the pCAMBIA1300-nLUC vector⁴⁷ to generate the corresponding constructs. For luciferase assay, the promoter of BRC1 was cloned into the pGreenII0800-LUC vector (Biovector, USA) at the SalI restriction site to produce the pBRC1::LUC construct. All these constructs were introduced into the A. tumefaciens strain EHA105.

To generate the FHY3 and BRC1 overexpression constructs, the full-length coding sequences of FHY3 and BRC1 were cloned into the pCAMBIA1301 vector (Biovector) to produce the FHY3-OE and BRC1-OE constructs, respectively. For rSPL9 overexpression, a miR156-resistant form of SPL9⁴⁸ without stop codon was cloned into the pSPYCE vector at the SalI restriction site to generate the rSPL9-OE construct. For SMXL6D (a nondegradable mutant form of SMXL6¹¹) overexpression, SMXL6D was constructed into the pSPYNE vector.

To generate the smxl6/7/8 triple knockout mutant, the CRISPR/Cas9 system was adopted following the method described by Zhao et al⁴⁹. In brief, potential target sites on the exons of SMXL6, SXML7, and SMXL8 were analyzed and selected using the SnapGene^® Viewer 3.2 software by the criteria of 5′-GG-(N)18-NGG-3′. The different forward primers sgR-F1 and sgR-F2 were paired with the reverse primer sgR-R to produce sgRNA1 and sgRNA2 fragments. Then these sgRNA fragments driven by the Arabidopsis ubiquitin U6-1 promoter were cloned into the CPB vector⁴⁹.

To generate the pBRC1wt::gBRC1 construct which harboring the BRC1 genomic coding region driven by the WT BRC1 promoter, the fragment containing the BRC1 promoter and the coding region was amplified from WT genomic DNA and ligated into the pCAMBIA1300 vector (Biovector) at the EcoRI/HindIII restriction sites. The pBRC1m::gBRC1 construct was produced by replacing the WT BRC1 promoter with the mutated promoter which is amplified from the pBRC1wt::gBRC1 construct in which the two SBP-binding sites GTAC were mutated into TTTT via site-directed mutagenesis.

All sequence confirmed transforming vectors were introduced into A. tumefaciens strain GV3101 and further transformed into WT Arabidopsis or specified backgrounds using the floral dip method⁵⁰. For overexpression vectors, more than 20 independent transgenic lines for each transformation vector were obtained and 3–5 representative lines (T₃ generation, homozygous plants) of each vector were selected for gene expression and phenotyping assays. For the smxl6/7/8 CRISPR knockout mutant, 27 independent transgenic lines were obtained and one homozygous line with mutated SMXL6/7/8 (T₃ generation) was selected and used for phenotyping assays. For pBRC1wt::gBRC1 and pBRC1m::gBRC1 constructs, more than ten independent transgenic lines of T₁ generation were used for phenotypic and gene expression assays.

All the primers used for plasmid construction are shown in Supplementary Table 1.

RNA extraction and gene expression analysis

For gene expression assay, tissues were frozen in liquid nitrogen and total RNA was extracted using the Trizol reagent (Invitrogen, USA). For total RNA extraction, axillary buds were harvested for BRC1 and leaves from 3-week-old plants for FHY3, SPL9, or SMXL6. The cDNA was synthesized using the FastQuant RT Kit (Tiangen Biotech, China) and the expression levels were detected using the Transstart Tip Green qPCR Super Mix (TransGen Biotech, China) on the Applied Biosystems Q3 real-time PCR system according the manufacturers’ instruction. The PP2A gene (AT1G13320) was used as an internal control and the relative transcript levels were calculated using the comparative Ct method⁵¹.

Y1H, Y2H, BiFC, and LCI assays

For Y1H assay, the AD-SPL and 2μ-pBRC1 or 2μ-pBRC2 plasmids were co-transformed into the yeast strain EGY48 as described in Lin et al.²⁷. For Y2H, the various plasmids for LexA-SPLs and AD-FHY3 or AD-SMXLs were co-transformed into the yeast strain EGY48 plus H18 and grown on the SD/-Ura/-Trp/-His medium. The positive transformants were further selected on the medium containing raffinose, galactose, and 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (Amresco, USA) as the substrate for blue color development.

For BiFC, the A. tumefaciens stain EHA105 harboring the pSPYCE-SPLs or pSPYNE-FHY3, FAR1, or SMXLs constructs were grown at 28 °C for 2–3 days. Then the positive transformants were cultured in liquid medium and the overnight cultures were diluted to OD₆₀₀ of 0.5 in a resuspension buffer containing 10 mM MgCl₂, 10 mM MES, and 100 mM acetosyringone and syringe-infiltrated into 4-week-old N. benthamiana leaves. The fluorescence within the infiltrated regions was visualized after 48 h using a Zeiss 510 Meta confocal laser scanning microscope.

For LCI, the A. tumefaciens stain EHA105 harboring pCAMBIA1300-cLUC-SPLs or pCAMBIA1300-nLUC-FHY3, FAR1, or SMXLs constructs were cultured in liquid medium at 28 °C for 2–3 days. After dilution with the resuspension buffer above, the cultures were syringe-injected into N. benthamiana leaves and incubated at 25 °C for 2–3 days. For the luciferase luminescence imaging, the infiltrated regions of the leaves were sprayed with 20 mg mL⁻¹ potassium luciferin (Gold Biotech, USA) and the luciferase luminescence was imaged using Night SHADE LB 985 system (Berthold, Germany).

Luciferase activity assay

The luciferase activity assay was performed following the method of Xie et al²⁴. Briefly, N. benthamiana leaves were co-injected with 35S::SPLs-SPYCE and pBRC1::LUC and incubated at 25 °C for 2–3 days. Then the injected leaves were detached and sprayed with 2 mg mL⁻¹ potassium luciferin (Gold Biotech, USA). The luciferase luminescence from the infiltrated area was imaged using Night SHADE LB 985 system (Berthold, Germany).

Gel mobility shift assay

The direct binding of SPL9 and SPL15 to the BRC1 promoter was detected using an EMSA kit (Beyotime, China) following the manufacturer’s protocol with probes listed in Supplementary Table. The GST and MBP proteins were used as the controls.

Recombinant protein production

For protein expression and purification, the SPL fragment containing SBP domain amplified from AD-SPLs above was cloned into the pGEX-4T-1 vector at the EcoRI restriction site to generate the GST-SPL9SBP and GST-SPL15SBP construct, respectively. The FHY3 fragment FHY3-I (containing the first 251 amino acids) and the full-length SMXL6 was cloned into the pET28a vector to generate the 6 × His-FHY3-I and 6 × His-SMXL6 constructs. All the constructs were transformed into the Escherichia coli strain Transette (TransGen Biotech, China). For recombinant protein expression, the bacteria strains were incubated in Luria-Bertain (LB) medium at 37 °C overnight and diluted at 1:100 with fresh LB medium and incubated at 37 °C for about 3 h until an optical density of 600 nm of 0.6–0.8 was reached. These bacteria were kept in the ice to cool down. Then the recombinant proteins GST-SPL9SBP, GST-SPL15SBP, or 6 × His-FHY3-I were induced by 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 16 °C overnight with gentle rotation (150 rpm), while the fusion proteins 6 × His-SMXL6 were induced by 0.1 mM IPTG at 4 °C for more than 20 h with gentle shaking (80 rpm). The cells were harvested by centrifugation at 3214 × g for 10 min at 4 °C. Then the pellets were resuspended in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO4, pH 7.4) for GST tag proteins or HIS Binding Buffer (250 mM KCl, 5 mM imidazole, 25 mM Tris-Cl, pH 8.0) for His tag proteins, respectively. After being ultrasonicated, the supernatant of cell debris was recovered by centrifugation at 13,000 g for 60 min. For GST tagged recombinant proteins, the supernatant was applied onto glutathione sepharose resin (GE Healthcare, USA) according to the manufacturer’s protocol. After extensive washing with PBS buffer, the GST fusion proteins were eluted with GST Elution Buffer (20 mM reduced glutathione, 100 mM Tris-Cl, pH 8.0). For His tagged fusion proteins, the supernatant was applied onto Ni-NTA His-Bind resin (Novagen, USA) following the manufacturers’ instruction. After extensive washing with HIS Binding Buffer, the His tagged fusion proteins were eluted with HIS Elution Buffer (250 mM KCl, 200 mM imidazole, 25 mM Tris-Cl, pH 8.0). These eluted proteins were then dialyzed against dialysis buffer (5 mM Tris-Cl, pH 8.0, 50 mM KCl), concentrated by ultrafiltration, aliquoted, frozen in liquid nitrogen and stored at −80 °C. The LC/MS/MS and Q-TOF analysis and the western blot of these proteins were provided in Supplementary Figs. 22 and 23 and Supplementary data set.

ChIP-qPCR assay

Eight-day-old transgenic seedlings of 35S::SPL9-HA or 35S::SPL15-HA were cross-linked with 1% formaldehyde and ground in liquid nitrogen. The chromatin complex was prepared following the method of Xie et al.²⁴. In brief, the supernatant was pre-cleared with 40 μl Protein-A-Agarose (16-157, EMD Millipore Corp.) and incubated at 4 °C for 1 h. Then the supernatant was moved into a microtube and 5 μl HA tag monoclonal antibodies (CB100005M, Cali-Bio) were added. After incubation at 4 °C for overnight with gentle agitation, 40 μl Protein-A-Agarose was added and incubated for 2 h. After washing, the immuno complex was eluted from the agarose beads. The precipitated DNA was then recovered and quantified using quantitative PCR with their individual primer pairs. The values were standardized to the input DNA to obtain the enrichment fold. PP2A was used as the internal control.

Western blotting analysis

For western blotting, 8-day-old seedlings were ground in liquid nitrogen and homogenized in extraction buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 10% glycerol, 0.1% Tween-20, 1 mM DTT, 1 mM phenylmethylsulphonyl fluoride, and complete protease inhibitor cocktail). After spinning, the supernatant was collected and the total protein quantified using the Bradford method. The purified anti-FLAG antibodies (M185-7, MBL, Japan) with 1:5000 dilution were used to detect FHY3 or FAR1 proteins. For the actin internal control, the plant actin monoclonal antibodies (BE7008, EASYBIO, China) at a dilution of 1:5000 were used. The western blotting was imaged with the Celvin S 420 Chemiluminescence Imaging System (Biostep, German).

Pull-down and Co-IP assay

For pull-down assay, the purified GST-SPL9SBP or GST-SPL15SBP fusion protein was added to the total protein extract of His-FHY3-I or His-SMXL6, and then incubated for 1 h at 4 °C under rotation. After washing with the protein extract buffer for three times, the samples were boiled with loading buffer and run on 10% SDS-PAGE gels for separation. The anti-GST antibodies (PM013-7, MBL, Japan) and anti-His antibodies (D291-7, MBL, Japan) with 1:5000 dilution were used to detect the protein level of SPL-SBP and FHY3-I/SMXL6, respectively.

For Co-IP, N. benthamiana leaves infiltrated with Agrobacterium harboring the constructs SPL9/15-MYC and FHY3/SMXL6-HA were collected and homogenized in 2 mL of co-IP buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 2 mM DTT, 0.1% Tween-20, 1 mM PMSF, and complete protease inhibitor cocktail). After centrifugation at 12,000 g for two times (10 min each time), the supernatant was collected and incubated for 2 h at 4 °C with anti-HA magnetic agarose beads (M180-10, MBL, Japan). After washing for three times with 1 mL of Co-IP buffer, the beads were boiled with loading buffer and run on 10% SDS-PAGE gels for separation. For western blotting, anti-MYC antibodies (M047-7, MBL, Japan), and anti-HA antibodies (561-7, MBL, Japan) with 1:5000 dilution were used to detect the levels of SPL9/15 or FHY3/SMXL6 proteins, respectively.

Measurement of relative fluorescence

The average fluorescence pixel intensity of each root was determined using Image J software (http://rsbweb.nih.gov/ij/; version 1.38) as described by Ye et al.^52,53. In brief, draw a close freehand line using the “straight” tool in the software along the outline of individual root and then do “subtract background” and calculate the average fluorescence using the “measure” tool.

Statistical analysis

All real-time PCR and other quantitative analysis were repeated at least three times. To evaluate the significant difference among genotypes treated with or without EOD-FR, the method of two-way analysis of variance with interaction was adopted by using SPSS software (version IBM SPSS Statistics 22.0). The least significance difference (LSD) test was adopted to analyze the significant differences among multiple groups. The Student t test was used to analyze the significant difference between two groups. The graphs with black dots and bars were produced using GraphPad Prism software (version 7.04) by entering the raw data.

Data availability

All data supporting the findings of this study are included within the article and its Supplementary Information files or available upon request from the authors. The source data for Figs. 1b–e, 2b–e, 3c–d, 4b–d, f, 5b–e, 6b, e and Supplementary Figs. 1–5, 7–9, 11 and 12, 14, 16, 18, 23 are provided in the Source Data file.

References

Domagalska, M. A. & Leyser, O. Signal integration in the control of shoot branching. Nat. Rev. Mol. Cell Biol. 12, 211–221 (2011).
Article CAS PubMed Google Scholar
Doebley, J., Stec, A. & Gustus, C. Teosinte branched1 and the origin of maize: evidence for epistasis and the evolution of dominance. Genetics 141, 333–346 (1995).
CAS PubMed PubMed Central Google Scholar
Takeda, T. et al. The OsTB1 gene negatively regulates lateral branching in rice. Plant J. 33, 513–520 (2003).
Article CAS PubMed Google Scholar
Minakuchi, K. et al. FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice. Plant Cell Physiol. 51, 1127–113 (2010).
Article CAS PubMed PubMed Central Google Scholar
Aguilar-Martínez, J. A., Poza-Carrión, C. & Cubas, P. Arabidopsis BRANCHED1 acts as an integrator of branching signals within axillary buds. Plant Cell 19, 458–472 (2007).
Article PubMed PubMed Central CAS Google Scholar
Gomez-Roldan, V. et al. Strigolactone inhibition of shoot branching. Nature 455, 189–194 (2008).
Article ADS CAS PubMed Google Scholar
Leyser, O. Strigolactones and shoot branching: a new trick for a young dog. Dev. Cell 15, 337–338 (2008).
Article CAS PubMed Google Scholar
Wang, Y. et al. Strigolactone/MAX2-induced degradation of brassinosteroid transcriptional effector BES1 regulates shoot branching. Dev. Cell 27, 681–688 (2013).
Article CAS PubMed Google Scholar
Jiang, L. et al. DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 504, 401–405 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhou, F. et al. D14-SCF (D3)-dependent degradation of D53 regulates strigolactone signalling. Nature 504, 406–410 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Wang, L. et al. Strigolactone signaling in Arabidopsis regulates shoot development by targeting D53-like SMXL repressor proteins for ubiquitination and degradation. Plant Cell 27, 3128–3142 (2015).
Article CAS PubMed PubMed Central Google Scholar
Wang, H. & Wang, H. The miR156/SPL module, a regulatory hub and versatile toolbox, gears up crops for enhanced agronomic traits. Mol. Plant 8, 677–688 (2015).
Article CAS PubMed Google Scholar
Wei, H., Zhao, Y., Xie, Y. & Wang, H. Exploiting SPL genes to improve maize plant architecture tailored for high-density planting. J. Exp. Bot. 69, 4675–4688 (2018).
CAS PubMed Google Scholar
Schwarz, S., Grande, A. V., Bujdoso, N., Saedler, H. & Huijser, P. The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis. Plant Mol. Biol. 67, 183–195 (2008).
Article CAS PubMed PubMed Central Google Scholar
Wang, J. W., Schwab, R., Czech, B., Mica, E. & Weigel, D. Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana. Plant Cell 20, 1231–1243 (2008).
Article CAS PubMed PubMed Central Google Scholar
Jiao, Y. et al. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat. Genet. 42, 541–544 (2010).
Article CAS PubMed Google Scholar
Miura, K. et al. OsSPL14 promotes panicle branching and higher grain productivity in rice. Nat. Genet. 42, 545–549 (2010).
Article CAS PubMed Google Scholar
Lu, Z. et al. Genome-wide binding analysis of the transcription activator ideal plant architecture1 reveals a complex network regulating rice plant architecture. Plant Cell 25, 3743–3759 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Song, X. et al. IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice. Cell Res. 27, 1128–1141 (2017).
Article CAS PubMed PubMed Central Google Scholar
Liu, J., Cheng, X., Liu, P. & Sun, J. miR156-targeted SBP-box transcription factors interact with DWARF53 to regulate TEOSINTE BRANCHED1 and BARREN STALK1 expression in bread wheat. Plant Physiol. 174, 1931–1948 (2017).
Article CAS PubMed PubMed Central Google Scholar
Ballaré, C. L. Keeping up with the neighbor: phytochrome sensing and other signaling mechanisms. Trends Plants Sci. 4, 97–102 (1999).
Article Google Scholar
Franklin, K. A. & Whitelam, G. C. Phytochromes and shade-avoidance responses in plants. Ann. Bot. 96, 169–175 (2005).
Article CAS PubMed PubMed Central Google Scholar
González-Grandío, E., Poza-Carrión, C., Sorzano, C. O. & Cubas, P. BRANCHED1 promotes axillary bud dormancy in response to shade in Arabidopsis. Plant Cell 5, 834–850 (2013).
Article CAS Google Scholar
Xie, Y. et al. Phytochrome-interacting factors directly suppress MIR156 expression to enhance shade-avoidance syndrome in Arabidopsis. Nat. Commun. 8, 348 (2017).
Article ADS PubMed PubMed Central CAS Google Scholar
Hudson, M., Ringli, C., Boylan, M. T. & Quail, P. H. The FAR1 locus encodes a novel nuclear protein specific to phytochrome A signaling. Genes Dev. 13, 2017–2027 (1999).
Article CAS PubMed PubMed Central Google Scholar
Wang, H. & Deng, X. W. Arabidopsis FHY3 defines a key phytochrome A signaling component directly interacting with its homologous partner FAR1. EMBO J. 21, 1339–1349 (2002).
Article CAS PubMed PubMed Central Google Scholar
Lin, R. et al. Transposase-derived transcription factors regulate light signaling in Arabidopsis. Science 318, 1302–1305 (2007).
Article ADS CAS PubMed PubMed Central Google Scholar
Stirnberg, P., Zhao, S., Williamson, L., Ward, S. & Leyser, O. FHY3 promotes shoot branching and stress tolerance in Arabidopsis in an AXR1-dependent manner. Plant J. 71, 907–920 (2012).
Article CAS PubMed Google Scholar
Smith, H. Light quality, photoperception, and plant strategy. Ann. Rev. Plant Physiol. 33, 481–518 (1982).
Article CAS Google Scholar
Li, G. et al. Coordinated transcriptional regulation underlying the circadian clock in Arabidopsis. Nat. Cell Biol. 13, 616–622 (2011).
Article CAS PubMed Google Scholar
Lin, R. et al. Discrete and essential roles of the multiple domains of Arabidopsis FHY3 in mediating phytochrome A signal transduction. Plant Physiol. 148, 981–992 (2008).
Article CAS PubMed PubMed Central Google Scholar
Toh, S., McCourt, P. & Tsuchiya, Y. HY5 is involved in strigolactone- dependent seed germination in Arabidopsis. Plant Signal Behav. 7, 556–558 (2012).
Article CAS PubMed PubMed Central Google Scholar
Jia, K. P., Luo, Q., He, S. B., Lu, X. D. & Yang, H. Q. Strigolactone-regulated hypocotyl elongation is dependent on cryptochrome and phytochrome signaling pathways in Arabidopsis. Mol. Plant 7, 528–540 (2014).
Article CAS PubMed Google Scholar
Shen, H., Luong, P. & Huq, E. The F-box protein MAX2 functions as a positive regulator of photomorphogenesis in Arabidopsis. Plant Physiol. 145, 1471–1483 (2007).
Article ADS CAS PubMed PubMed Central Google Scholar
Shen, H., Zhu, L., Bu, Q. Y. & Huq, E. MAX2 affects multiple hormones to promote photomorphogenesis. Mol. Plant 5, 750–762 (2012).
Article PubMed CAS Google Scholar
Koltai, H. et al. A tomato strigolactone-impaired mutant displays aberrant shoot morphology and plant interactions. J. Exp. Bot. 61, 1739–1749 (2010).
Article CAS PubMed PubMed Central Google Scholar
Nagata, M. et al. Red/far red light controls arbuscular mycorrhizal colonization via jasmonic acid and strigolactone signaling. Plant Cell Physiol. 56, 2100–2109 (2015).
CAS PubMed Google Scholar
Yu, S. et al. Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMSA promoter binding-like transcription factors. Plant Cell 24, 3320–3332 (2012).
Article CAS PubMed PubMed Central Google Scholar
Warnasooriya, S. N. & Brutnell, T. P. Enhancing the productivity of grasses under high-density planting by engineering light responses: from model systems to feed stocks. J. Exp. Bot. 65, 2825–2834 (2014).
Article PubMed Google Scholar
Duvick, D. N. The contribution of breeding to yield advances in maize (Zea mays L.). Adv. Agron. 86, 83–145 (2005a).
Article Google Scholar
Duvick, D. N. Genetic progress in yield of United States maize (Zea mays L.). Maydica 50, 193–202 (2005b).
Google Scholar
Sawers, R. J., Sheehan, M. J. & Brutnell, T. P. Cereal phytochromes: targets of selection, targets for manipulation? Trends Plant Sci. 10, 138–143 (2005).
Article CAS PubMed Google Scholar
Kebrom, T. H., Brutnell, T. P. & Finlayson, S. A. Suppression of sorghum axillary bud outgrowth by shade, phyB and defoliation signalling pathways. Plant Cell Environ. 33, 48–58 (2010).
CAS PubMed Google Scholar
Kebrom, T. H., Burson, B. L. & Finlayson, S. A. Phytochrome B represses Teosinte Branched1 expression and induces sorghum axillary bud outgrowth in response to light signals. Plant Physiol. 140, 1109–1117 (2006).
Article CAS PubMed PubMed Central Google Scholar
Whipple, C. J. et al. grassy tillers1 promotes apical dominance in maize and responds to shade signals in the grasses. Proc. Natl Acad. Sci. USA 108, E506–E512 (2011).
Article CAS PubMed PubMed Central Google Scholar
Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438 (2004).
Article CAS PubMed Google Scholar
Chen, H. et al. Firefly luciferase complementation imaging assay for protein–protein interactions in plants. Plant Physiol. 146, 368–376 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Wu, G. et al. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138, 750–759 (2009).
Article CAS PubMed PubMed Central Google Scholar
Zhao, Y. et al. An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. Sci. Rep. 6, 23890 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Article CAS PubMed Google Scholar
Schmittgen, T. & Livak, K. Analysis of relative gene expression data using RT-qPCR. Methods 25, 402–408 (2001).
Article PubMed CAS Google Scholar
Soundappan, I. et al. SMAX1-LIKE/D53 family members enable distinct MAX2-dependent responses to strigolactones and karrikins in Arabidopsis. Plant Cell 27, 3143–3159 (2015).
Article CAS PubMed PubMed Central Google Scholar
Ye, J. et al. Arabidopsis formin3 directs the formation of actin cables and polarized growth in pollen tubes. Plant Cell 21, 868–884 (2009).
Article CAS Google Scholar

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Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (31770210 and 31570191) and National Key Research and Development Program of China (2016YFD0100303).

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Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Yurong Xie, Yang Liu, Mengdi Ma & Baobao Wang
Graduate School of Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Qin Zhou, Yongping Zhao & Binbin Zhao
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, 510642, Guangzhou, China
Hongbin Wei & Haiyang Wang
Guangdong Laboratory for Lingnan Modern Agriculture, 510642, Guangzhou, China
Haiyang Wang

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Yurong Xie
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H.Wang and Y.X. designed the research; Y.X., Y.L., M.M., Q.Z., Y.Z., B.Z., B.W. and H.Wei performed the research and analyzed the data; H.Wang and Y.X. wrote the paper.

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Xie, Y., Liu, Y., Ma, M. et al. Arabidopsis FHY3 and FAR1 integrate light and strigolactone signaling to regulate branching. Nat Commun 11, 1955 (2020). https://doi.org/10.1038/s41467-020-15893-7

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Received: 22 June 2019
Accepted: 01 April 2020
Published: 23 April 2020
DOI: https://doi.org/10.1038/s41467-020-15893-7

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