A Critical Role of the Soybean Evening Complex in the Control of Photoperiod Sensitivity and Adaptation

A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation

Tiantian Bua,1, Sijia Lua,1, Kai Wanga,1, Lidong Donga,1, Shilin Lib, Qiguang Xieb, Xiaodong Xub, Qun Chenga, Liyu Chena, Chao Fanga, Haiyang Lia, Baohui Liua,c, James L. Wellerd, and Fanjiang Konga,c,2

aInnovative Center of Molecular Genetics and Evolution, School of Life Sciences, Guangzhou University, 510006 Guangzhou, China; bState Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, 475004 Kaifeng, China; cThe Innovative Academy of Seed Design, Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 150081 Harbin, China; and dSchool of Natural Sciences, University of Tasmania, Hobart, 7001 TAS, Australia

Edited by Xinnian Dong, Duke University, Durham, NC, and approved December 22, 2020 (received for review May 21, 2020) Photoperiod sensitivity is a key factor in plant adaptation and crop network, the evening complex (EC)—comprising EARLY production. In the short-day plant soybean, adaptation to low lat- FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4), itude environments is provided by mutations at the J locus, which and LUX ARRHYTHMO (LUX)—is a transcriptional repres- confer extended flowering phase and thereby improve yield. The sor complex and a core component of the plant circadian clock identity of J as an ortholog of Arabidopsis ELF3, a component of (5). LUX is a single MYB domain-containing SHAQYF-type the circadian evening complex (EC), implies that orthologs of other GARP transcription factor, and appears to mediate the inter- EC components may have similar roles. Here we show that the two action of the EC with target promoters through direct binding of soybean homeologs of LUX ARRYTHMO interact with J to form a the MYB domain through a specific LUX binding site (LBS) soybean EC. Characterization of mutants reveals that these genes motif GATWCG (where W indicates A or T) (6, 7). Recent are highly redundant in function but together are critical for flow- studies indicate that a prion-like domain in ELF3 functions as a lux1 lux2 ering under short day, where the double mutant shows thermosensor, while ELF4 can stabilize the function of ELF3 (8, extremely late flowering and a massively extended flowering 9). EC directly regulates multiple clock output pathways, such as phase. This phenotype exceeds that of any soybean flowering mu- “ hypocotyl growth, flowering, defense, and leaf senescence (6, tant reported to date, and is strongly reminiscent of the Maryland 10–13). The importance of the EC for flowering-time control and Mammoth” tobacco mutant that featured in the seminal 1920 AGRICULTURAL SCIENCES photoperiodism in particular is indicated by the fact that Arabi- study of plant photoperiodism by Garner and Allard [W. W. Gar- dopsis mutants affecting any one of its three components have ner, H. A. Allard, J. Agric. Res. 18, 553–606 (1920)]. We further markedly impaired photoperiod responsiveness (14–16), and a demonstrate that the J–LUX complex suppresses transcription of E1 growing list of flowering-time variants in crop species have also the key flowering repressor and its two homologs via LUX – binding sites in their promoters. These results indicate that the been linked to EC components (17 26). EC–E1 interaction has a central role in soybean photoperiod sensi- Soybean [Glycine max (L). Merr.] is a major legume crop that tivity, a phenomenon also first described by Garner and Allard. EC produces protein and oil and provides more than a quarter of the ’ and E1 family genes may therefore constitute key targets for cus- world s protein for food and animal feed (27). Cultivated soy- tomized breeding of soybean varieties with precise flowering time bean was domesticated from its wild relative (Glycine soja Sieb. adaptation, either by introgression of natural variation or gener- & Zucc.) more than 5,000 y ago in temperate regions of China ation of new mutants by gene editing. Significance flowering | adaptation | LUX ARRHYTHMO (LUX) | evening complex (EC) | soybean In many plant species, the timing of flowering is sensitive to photoperiod. In many crop species, genetic variation in this t is now widely appreciated that the timing of flowering, and sensitivity is critical for adaptation to specific regions and Ithe extent to which it is responsive to environmental cues, is management practices. This study identifies a component of one of the most important determinants of crop adaptation and the genetic pathway controlling flowering time in soybean, a yield (1). One hundred years ago, Wightman Garner and Harry legume crop of major global importance. Notably, plants lack- Allard (2) made the first comprehensive report on plant pho- ing this component flower extremely late. Photoperiod sensi- toperiodism in a seminal paper, which prominently featured tivity in plants, including soybean, was first systematically soybean and tobacco as model plants. Over the following de- described in a seminal paper 100 y ago, and the results pre- cades the physiological and molecular basis of this phenomenon sented here establish an important new molecular step un- has been investigated and characterized in detail, first in Arabi- derlying this response. This step is a critical control point that could be genetically adjusted to engineer photoperiod sensi- dopsis, and increasingly in other species. Although our under- tivity for yield improvement across a broad range of locations standing of the molecular diversity in mechanisms of flowering- and agricultural contexts. time regulation is still relatively limited, certain fundamental

features are proving to be widespread, if not universal. In the Author contributions: B.L. and F.K. designed research; T.B., S. Lu, K.W., L.D., S. Li, Q.C., case of photoperiodism, it appears that an interaction between L.C., C.F., and H.L. performed research; T.B., Q.X., and X.X. analyzed data; and T.B., J.L.W., light perception and endogenous circadian rhythms directs the and F.K. wrote the paper. photoperiod-specific expression of genes in the florigen family, The authors declare no competing interest. many of which encode mobile signals that move from leaf and This article is a PNAS Direct Submission. shoot apex to induce flowering. Published under the PNAS license. The genetic network responsible for the generation of circa- 1T.B., S. Lu, K.W., and L.D. contributed equally to this work. dian rhythms (the circadian “clock”) is understood to consist of 2To whom correspondence may be addressed. Email: [email protected]. – multiple interlocked transcriptional translational feedback This article contains supporting information online at https://www.pnas.org/lookup/suppl/ loops, and dozens of genes constituting these loops have been doi:10.1073/pnas.2010241118/-/DCSupplemental. identified in the model plant Arabidopsis (3, 4). Within this Published February 8, 2021.

PNAS 2021 Vol. 118 No. 8 e2010241118 https://doi.org/10.1073/pnas.2010241118 | 1of10 Downloaded by guest on September 25, 2021 between 32° and 40°N (28–30). As first demonstrated by Garner (40). E3 and E4 encode phytochrome A (PHYA) genes, PHYA3 and Allard (2), soybean is a short-day (SD) plant (SDP) and, and PHYA2, respectively (41, 42), while E9 and E10 have been whereas the wild ancestor and many primitive forms of domes- identified as florigen genes FT2a and FT4, respectively (35, 43). ticated soybean are strongly photoperiod-sensitive, modern soy- Recently, the Tof11 and Tof12 loci were identified as orthologs bean cultivars vary widely in their degree of sensitivity. This has of another Arabidopsis circadian clock component PRR3, and enabled adaptation of the crop across a wide latitudinal range shown to have contributed to the development of early flowering from 50°N to 35°S, and reflects natural variation in genes con- and latitudinal adaptation during early evolution of domesti- trolling flowering and maturity, with allelic combinations speci- cated soybean (33, 44). fying optimal adaptation to narrow latitudinal zones (31). A Recent efforts have also focused on genes conferring adap- growing number of loci contributing to this adaptation have been tation to low latitudes. When commercial soybean varieties de- identified and characterized to the molecular level, including the veloped in temperate regions are grown at lower latitudes, they E series (E1–E11), Tof11, Tof12, and J (31–37). Among these, the legume-specific E1 gene has emerged as a mature undesirably early and have extremely poor grain yield. major regulator of the photoperiod response and a critical point This limitation was overcome in the 1970s with the introduction of integration within the soybean flowering pathway (33, 34, 38, of the long-juvenile (LJ) trait, which extends the vegetative phase 39). E1 encodes a B3 superfamily member, which is itself and improves yield under SD conditions, enabling productive strongly regulated by photoperiod and controls flowering cultivation in tropical regions (45–49). A major locus conferring through its repressive effects on expression of two key FT genes, this trait, J, is now known to be an ortholog of the Arabidopsis EC FT2a and FT5a (39). E2 has been identified as an ortholog of component ELF3 (34). Functional characterization of J has GIGANTEA (GI), a component of Arabidopsis circadian clock provided a working model for its role in SD flowering in which it

A C D

Fig. 1. Protein interactions of soybean EC (SEC). (A) J interacts with LUX1 and LUX2 in yeast. Yeast cells transformed with indicated genes were selected on DDO (lacking Leu and Trp) and QDO (lacking Ade, His, Leu, and Trp) media. (B) J interacts with LUX1 and LUX2 in Nicotiana benthamiana leaves in a BiFC assay. LUX1 and LUX2 were fused to the N terminus of YFP and J was fused to the C terminus of YFP. The constructs were coinjected into N. benthamiana leaves, and YFP signals were observed after 48 to 72 h. (Scale bars, 20 μm.) Three biological replicates were performed. (C) LUX1 and LUX2 can pull down J. MBP, MBP-LUX1, and MBP-LUX2 proteins were expressed in Escherichia coli, and J-His protein was expressed using an in vitro translation system. Purified proteins were used for the pull-down assay. MBP, MBP-LUX1, and MBP-LUX2 were detected with anti-MBP antibody, and J-His protein was detected with anti- His antibody. (D) LUX1 and LUX2 interact with each other and themselves in yeast. Yeast cells transformed with indicated genes were selected on DDO and QDO media. (E) LUX1 and LUX2 interact with each other and themselves in N. benthamiana leaves in a BiFC assay. LUX1 and LUX2 were fused to the N and C terminus of YFP. The constructs were coinjected into N. benthamiana leaves, and YFP signals were observed after 48 to 72 h. (Scale bars, 20 μm.) Three biological replicates were performed.

2of10 | PNAS Bu et al. https://doi.org/10.1073/pnas.2010241118 A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation Downloaded by guest on September 25, 2021 acts to repress E1 transcription, thereby relieving the E1 sup- Protein Interactions of LUX1, LUX2, and J. To test whether either of pression of FT2a and FT5a, and promoting flowering (34). the two LUX proteins might interact physically with J, we first Although J influences flowering through transcriptional re- performed yeast two-hybrid assays (Y2H), which showed that pression of E1 (34), there is no direct evidence for how this is both LUX1 and LUX2 indeed interact with J (Fig. 1A). These achieved. However, the evidence from Arabidopsis implies that J interactions were confirmed using in vitro pull-down assays may participate in a complex that is targeted to promoters of (Fig. 1C), and extended by bimolecular fluorescence comple- various target genes through the direct DNA-binding activity of mentation (BiFC) assays, which additionally indicated that they LUX orthologs (6). In this study, we investigated the molecular occur in vivo and take place in the nucleus (Fig. 1B). It has been role of J through an examination of the two soybean LUX ho- reported that most transcription factors bind to target DNA mologs, LUX1 and LUX2. We demonstrate that both soybean sequences as dimers (56). We examined whether LUX1 and LUX proteins physically interact with J and directly bind to the LUX2 have this property using BiFC and Y2H assays. The results showed that both LUX1 and LUX2 could self-interact, and LBS in the E1 promoter to repress E1 expression. We also use also interact with each other (Fig. 1 D and E). Our results sug- targeted knockout of LUX genes to show that they play a re- gested that LUX1 and LUX2 might form a heterodimer and dundant but critical role in flowering, maturity, adaptation, and homodimer to directly regulate targeted genes. Taken together, yield and investigate their potential regulatory roles. Our results these results indicated that three proteins LUX1, LUX2, and J suggest that the soybean EC might function as an essential node interact with each other in the nucleus to form the EC in soybean to connecting circadian clock components and light signaling control development, flowering, maturity, adaptation, and yield. pathways to multiple target genes for control of flowering time and other responses. LUX1 and LUX2 Are Direct Transcriptional Repressors of E1. We Results previously showed that J is a transcriptional repressor of the key soybean flowering repressor E1, and binds directly to the E1 Characterization and Expression Patterns of LUX1 and LUX2 in Soybean. promoter (34). To investigate whether LUX1 and LUX2 might It is well documented that soybean has undergone two rounds of be similarly involved in the transcriptional repression of E1,we whole-genome duplication (WGD) after its divergence from the ∼ examined the molecular nature of the relationship between LUX Arabidopsis lineage: One occurred recently at 13 Mya after its split E1 Arabidopsis ∼ proteins and using an protoplast transient ex- from common bean (Phaseolus vulgaris)at 19 Mya, while the other pression assay. When a p35S:LUX1 or p35S:LUX2 construct was ∼ – ancient one occurred at 59 Mya (50 52). In the soybean reference cotransformed with a pE1:LUC construct (Fig. 2A), relative LUC ∼ AGRICULTURAL SCIENCES genome, 75% of the predicted genes exist as duplicated gene pairs activity was significantly suppressed to a similar extent as a derived from the ∼13-Mya WGD event, while the remaining 25% p35S:J construct (Fig. 2B), indicating that LUX1 and LUX2 have reverted to singletons (50, 53, 54). As expected, two homeol- protein might bind to the E1 promoter to suppress its activity. ogous LUX gene pairs deriving from the recent ∼13 Mya WGD When a p35S:J construct and a p35S:LUX construct were were identified in the Williams 82 (W82) reference genome (Phy- cotransformed with a pE1:LUC construct, relative LUC activity tozome; https://phytozome.jgi.doe.gov/soybean), and they are desig- was much lower (Fig. 2B), suggesting that J and LUX proteins nated as LUX1 (Glyma.12G060200) and LUX2 (Glyma.11G136600), could enhance the suppressive activities of each other in the J– as previously reported (34). Phylogenetic analysis of 40 LUX-like LUX complex. Two-way ANOVA revealed that relative LUC − proteins from 24 species showed that LUX1 and LUX2 clustered activity is suppressed by J (P = 3.8 × 10 17), LUX (P = 1.2 × − − together and fell into the same clade with other legume LUX pro- 10 19), and J × LUX (P = 3.9 × 10 9), indicating that the en- teins (SI Appendix,Fig.S1A). As a paleopolyploid species, there are hancement is synergistic. However, when both constructs of two LUX homologs in soybean, while LUX is a single-copy gene in LUX1 and LUX2 were put together, the relative LUC activity diploid legume species. Protein sequence alignment revealed that were not significantly lower than either of the single constructs, these LUX-like proteins were highly conserved in the DNA-binding suggesting that the functions of these genes have remained MYB domain (SI Appendix,Fig.S2). largely equivalent following their duplication during the poly- To analyze the expression characteristics of LUX1 and LUX2, ploidization. To confirm that LUX proteins could bind to the E1 regulation of their transcripts was investigated by qRT-PCR. As promoter in a soybean in vivo system, we performed chromatin an SDP, soybean plants flower when day length is shorter than immunoprecipitation (ChIP)-qPCR assays in p35S:LUX1-FLAG and p35S:LUX2-FLAG transgenic plants and W82 plants the maximum critical value, and the daylength regime for SD is (Fig. 2 C and D). Using a similar assay, we previously demon- generally 12-h light/12-h dark (2, 55). We examined the expres- strated the physical association of J with three regions of the E1 sion patterns of LUX under artificial SD (ASD) conditions (12-h promoter containing LBS (34). Our result showed that LUX1 light/12-h dark) in the growth chamber. The tissue-specific ex- and LUX2 were also associated with strong enrichment of E1 pression study showed that both LUX1 and LUX2 were promoter sequences around three of these same LBS, at −264 expressed in all tissues examined (SI Appendix, Fig. S1B). The bp, −799 bp, and −975 bp upstream of the ATG (Fig. 2 C and time-course expression study showed that the expression of both D). Finally, we performed EMSA to determine whether LUX1 genes in leaves increased continuously from 10 d after emer- and LUX2 proteins directly bind to E1 promoters in vitro. We gence (DAE) to 25 DAE and peaked at 25 DAE, then decreased found that LUX1 and LUX2 recombinant proteins could both from 25 DAE to 35 DAE (SI Appendix, Fig. S1D). Under SD directly bind to the LBS sites in the E1 promoters (Fig. 2 C, E, conditions, expression of both genes showed clear diurnal and F). These results indicate that LUX1 and LUX2 proteins rhythms and peaked at dusk (SI Appendix, Fig. S1F), consistent physically interact with J and likely allow the J–LUX complex to with the expression pattern of LUX in other species (15, 19, 20). directly bind to the E1 promoter and repress its activity. We also found that in the same samples, the expression of J, the soybean ortholog of Arabidopsis EC gene ELF3 (34), showed LUX1 and LUX2 Are the Critical Regulators in Soybean Flowering similar tissue-specific, time-course, and diurnal patterns to those under SD Conditions. To obtain direct genetic evidence for the of the two LUX genes (SI Appendix, Fig. S1 C, E, and G). function of LUX1 and LUX2 in soybean flowering time control, Therefore, we considered it likely that LUX1 and LUX2 might we used a CRISPR/Cas9-mediated genome-editing approach to interact physically with J to form EC in soybean, a complex generate the soybean knockout mutants for both genes. We se- similar to that in Arabidopsis (6). lected three genomic sites for simultaneous targeting of both

Bu et al. PNAS | 3of10 A critical role of the soybean evening complex in the control of photoperiod sensitivity and https://doi.org/10.1073/pnas.2010241118 adaptation Downloaded by guest on September 25, 2021 A B 0.04

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Fig. 2. LUX1 and LUX2 directly associate with the promoter of E1 to suppress its transcriptions. (A) Constructs of LUX1, LUX2, J, and E1 used for the transient expression assay in Arabidopsis protoplast. LUC, luciferase; REN, Renilla luciferase. (B) LUX1, LUX2, and J proteins suppress transcription from the E1 promoter in Arabidopsis protoplast. Values are shown as mean ± SD from three biological replicates. Different letters indicate significant differences by one-way ANOVA followed by Tukey’s post hoc test with SPSS statistics software. False-discovery rate (FDR)-adjusted P < 0.05. Two-way ANOVA revealed that the − − − relative LUC activity is suppressed by J (P = 3.8 × 10 17), LUX (P = 1.2 × 10 19), and J × LUX (P = 3.9 × 10 9). (C) Schematic of the E1 gene and regions tested for enrichment in the ChIP assay and binding in the EMSA assay. (D) ChIP of E1 amplicons using W82, p35S:LUX1-FLAG, and p35S:LUX2-FLAG. Values are shown as mean ± SD from three biological replicates. Different letters indicate significant difference among the samples using the same primer by one-way ANOVA followed by Tukey’s post hoc test with SPSS statistics software. FDR-adjusted P < 0.05. Capital letters compare with each other, and lowercase letters compare with each other. (E and F) EMSA detected binding of GST-LUX1 (E) and GST-LUX2 (F) protein to the LBS of the E1 promoter.

LUX1 and LUX2 coding sequences (SI Appendix, Fig. S3), aim- frame-shift mutation after the M16 codon in both genes. In ing to obtain lux1 and lux2 single mutants and lux1 lux2 double lux1 lux2-2, we found the same LUX1 mutation (lux1) but a mutants. Appropriate single-guide RNA (sgRNA)/Cas9 vectors deletion of 22 bases in the LUX2 coding region, which intro- were constructed and transformed into the soybean cultivar duced a frameshift after codon R146. All three mutations W82, ultimately generating one independent lux1 mutant line, specified a premature stop codon (SI Appendix, Fig. S3). two lux2 mutant lines, and two lux1 lux2 double-mutant lines Under flowering-inductive ASD conditions (12-h light/12-h dark), the LUX1 and LUX2 overexpression transgenic plants, (Table 1). In the lux1 lux2-1 mutant, there was a deletion of a p35S:LUX1-FLAG and p35S:LUX2-FLAG showed no significant single G in both LUX1 and LUX2 coding regions, which caused a effect on the flowering time (SI Appendix, Fig. S4 A and B), which may suggest the duplicated homeologous pairs already have been over the functional threshold dosages (57). However, Table 1. Homozygous mutants of LUX1 and LUX2 the double lux1 lux2-1 mutant showed an extreme delay, not LUX1 LUX2 flowering until nearly 100 DAE compared to the wild-type plants flowering at 23 DAE under natural SD (NSD, 13-h light/11-h Target 1 Target 2 Target 1 Target 2 dark) conditions in Guangzhou (23° 16′ N, 113° 23′ E), China. In contrast, the single lux1 and lux2-1 mutations showed no signif- lux1 −1bp ——— ——− — icant effect on flowering time (Figs. 3 and 4A). These results lux2-1 1bp show that LUX1 and LUX2 are functionally redundant but to- lux2-2 ———−22 bp − — − — gether play critical roles in regulation of soybean flowering. lux1 lux2-1 1bp 1bp Mutants carrying a second loss of function allele of LUX2, lux2- lux1 lux2-2 −1bp ——−22 bp 2, and lux1 lux2-2, showed flowering phenotypes consistent with

4of10 | PNAS Bu et al. https://doi.org/10.1073/pnas.2010241118 A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation Downloaded by guest on September 25, 2021 A B

C D AGRICULTURAL SCIENCES

Fig. 3. Phenotypes of lux1 and lux2 mutants. Phenotypes of wild-type plants (WT, W82) and homozygous mutants at 25 DAE (A), 95 DAE (B), 120 DAE (C), 155 DAE (D) under NSD (13-h light/11-h dark) conditions. Red box, magnified view. (Scale bars, 20 cm). The lux1 lux2-1 mutant, also called as Guangzhou Mammoth, continuously grows and keeps flowering, as shown in C and D.InC, Guangzhou Mammoth is 210-cm high and in D, it grows up to 250-cm high without stopping growing.

those associated with lux2-1 and lux1 lux2-1. (Fig. 4A and SI under both ASD (12-h light/12-h dark) and artificial long day Appendix, Fig. S5). Under these conditions, wild-type plants (ALD, 16-h light/8-h dark) conditions in a growth chamber. produce around 10 reproductive nodes on the main stem before Under ASD, the single lux1 or lux2-1 mutants showed no sig- undergoing proliferative arrest of the primary shoot meristem nificant difference with the wild-type plants, which flowered at 24 and entering monocarpic senescence at ∼15 wk of age. DAE, while the Guangzhou Mammoth flowered at about 77 In contrast, the lux1 lux2-1 mutant, in addition to first initi- DAE (SI Appendix, Fig. S6). Intriguingly, under ALD conditions, ating flowering 11 wk later than wild-type, also showed a dra- the Guangzhou Mammoth also flowered at about 78 DAE matically extended reproductive period, with the primary shoot compared to the wild-type plants that flowered at 51 DAE, while apex continuing to grow and produce axillary flowers for a fur- the single lux1 and lux2-1 mutations showed no significant dif- ther 22 wk without showing the stopping apical growth (Fig. 3 C ference with the wild-type plants (SI Appendix, Fig. S6), indi- and D). This massive extension of the growth period was ac- cating that EC also plays important roles in regulating soybean companied by a striking thickening of stems, giving plants the flowering under LD conditions and complete impairment of EC appearance of a small tree (Fig. 3 C and D). This dramatic effect abolishes soybean photoperiod sensitivity. Collectively, these results on growth habit is reminiscent of the famous photoperiod- suggest that EC plays critical roles in soybean photoperiod response. sensitive tobacco mutant Maryland Mammoth that was prom- inent in the seminal study of plant photoperiodism by Garner LUX1 and LUX2 Act Upstream of the Legume-Specific Flowering and Allard (2) and nicely illustrated by Amasino (58). We Repressors E1 and E1 Homologs. To further understand the func- therefore named this lux1 lux2-1 double mutant as “Guangzhou tional mechanisms underlying EC, we investigated E1 expression Mammoth” to pay homage to Maryland Mammoth. In addition, in lux1, lux2-1, lux1 lux2-1, and wild-type W82. Under SD con- we also evaluated the flowering time of Guangzhou Mammoth ditions, the expression of E1 in W82 was very low, as reported

Bu et al. PNAS | 5of10 A critical role of the soybean evening complex in the control of photoperiod sensitivity and https://doi.org/10.1073/pnas.2010241118 adaptation Downloaded by guest on September 25, 2021 0.25 ABW82 lux2-1 120 b lux1 lux1 lux2-1 b 0.20

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/ lux1 lux2-1 lux1 lux2-1 0.04 E1La E1Lb/ 0.02 0.02

0.00 0.00 04812162024 0 4 8 12162024 Zeitgeber time (h) Zeitgeber time (h)

Fig. 4. LUX1 and LUX2 redundantly regulate transcript abundance of the soybean core flowering genes E1 and FT.(A) Flowering time of W82 and homozygous mutants under NSD conditions (13-h light/11-h dark). Different letters indicate significant differences by Kruskal–Wallis one-way ANOVA followed by multiple-comparison test with SPSS statistics software. FDR-adjusted P < 0.05. The flowering time is shown as the mean values ± SD, n > 10 plants. (B–F) Diurnal expression of E1 (B), FT2a (C), FT5a (D), E1La (E), and E1Lb (F)inW82,lux1, lux2-1, and lux1 lux2-1 plants at 15 DAE under ASD (12-h light/12-h dark). Data shown relative to the control gene Tubulin and represent means ± SD for three biological replicates. The dashed line indicates nonlinear regression curve. Nonlinear regression analysis was performed by GraphPad Prism 8.

previously (39), but showed a massive derepression in lux1 lux2-1 consistent with the higher expression of E1 (Fig. 4B) and extremely double-mutant plants and a clear diurnal rhythm with a peak at late flowering phenotypes (Fig. 3). These results show that the two dusk (Fig. 4B). However, no obvious induction of E1 was ob- LUX genes act in a functionally redundant manner to fully suppress served in lux1 and lux2-1 single mutants (Fig. 4B), consistent with E1 expression, consequently relieving the repression of FT2a and their flowering-time phenotype (Figs. 3 and 4A), further sup- FT5a thereby promoting flowering and maturity and reducing porting the functional redundancy of LUX1 and LUX2, which overall yield potential under SD conditions. was well documented with the notion that duplicated genes were The soybean genome has two E1 homologs, E1-like-a (E1La) retained without functional changes to maintain their dosage and E1Lb, which were suggested to function similarly to, but to balance (57, 59, 60). To some extent, the functional redundancy some extent independently from E1 in the control of flowering could be explained by which the heterodimers and homodimers and adaptation (62, 63). We therefore examined the diurnal of LUX1 and LUX2 formed (Fig. 1 D and E) might play equivalent expression of E1La and E1Lb in W82, lux1, lux2-1, and lux1 lux2- roles in soybean EC in regulating flowering, which has been 1 under SD conditions. Both genes showed patterns of expres- reported in several cases (56). We also examined E1 expression in sion very similar to E1, with no significant expression in W82 and p35S:LUX1-FLAG and p35S:LUX2-FLAG plants; the result both single mutants, but a strong derepression with a peak at showed that E1 expression was not influenced by overexpression of ZT12 in lux1 lux2-1 (Fig. 4 E and F). As in the case of E1, the LUX1 and LUX2, which was consistent with the lack of effect on promoters of E1La and E1Lb also contained several LBS sites flowering phenotypes (SI Appendix,Fig.S4B and C). Previous implying that the EC could also directly bind to these sites to studies have shown that the central role of E1 in the photoperiod suppress E1La and E1Lb transcription (SI Appendix, Fig. S7A). regulation of soybean flowering largely reflected its repression of To examine whether LUX proteins could bind to the E1La and two key FT homologs, FT2a and FT5a (34, 39, 61). We further E1Lb promoter in soybean, we performed ChIP-qPCR assays in examined the expression of FT2a and FT5a and found that the p35S:LUX1-FLAG and p35S:LUX2-FLAG transgenic plants and transcriptions of both FT genes were nearly not detected in W82 plants. Our result showed that LUX1 and LUX2 were also lux1 lux2-1 double-mutant plants (Fig. 4 C and D), which is associated with strong enrichment of the E1La and E1Lb

6of10 | PNAS Bu et al. https://doi.org/10.1073/pnas.2010241118 A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation Downloaded by guest on September 25, 2021 promoter (SI Appendix, Fig. S7 B and C). The direct repressive Various Flowering-Associated Genes under Regulation by Soybean EC. roles of LUX1 and LUX2 on E1La and E1Lb were also tested in To gain further insight into the nature of the genes regulated by Arabidopsis protoplast transient expression assay, in which LUX1 the soybean EC, we performed transcriptome sequencing and LUX2 were capable of repressing the transcription from the (RNA-seq) on lux1, lux2-1, lux1 lux2-1, and W82 plants. Com- E1La and E1Lb promoter (SI Appendix, Fig. S7 D–F). These pared with W82, there were 2,297, 1,018, and 3,316 differentially results indicate that E1La and E1Lb are also negatively regu- expressed genes (DEGs) in leaves in lux1, lux2-1, and lux1 lux2-1 lated by LUX1 and LUX2, and provide further evidence that mutant plants (Dataset S1). Interestingly, at least 43 DEGs in E1La and E1Lb play a function similar to E1 in photoperiodic lux1 lux2-1 showed homology with known flowering time- induction of flowering in soybean. Overall, these results indicate associated genes from Arabidopsis by using phytozome and that full loss of LUX function (presumably causing complete Uniprot databases (SI Appendix, Fig. S8A and Dataset S2). evening complex impairment) releases expression of three of Among them, several DEG regulatory pathways involved in these E1 family genes, which are then able to repress FT ex- photoperiod, the gibberellic acid (GA) pathway, and circandian clocks were misregulated by LUX1 and LUX2. Consistent with pression and thereby generate extremely late-flowering pheno- – types under SD conditions. our qRT-PCR analysis (Fig. 4 B F), E1, E1La, and E1Lb were Taken together, these results propose a model that LUX1 and strongly up-regulated, and FT2a and FT5a were strongly re- FT LUX2 are functionally redundant in modulating photoperiod- pressed. Three other homologs were also significantly misregulated in lux1 lux2-1. Like FT2a, FT2b (Glyma.16G151000) regulated flowering in soybean under SD conditions, and both was also significantly down-regulated in lux1 lux2-1, implying that of them suppress expressions of E1 and its homologs by binding that FT2b may also function as a flowering promoter. In contrast, to the LBS in their promoters, which relieves the E1-dependent FT4 (Glyma.08G363100) and to a lesser extent FT1a (Gly- FT2a FT5a transcriptional repression of and , thereby promoting ma.18G298900) were substantially up-regulated in lux1 lux2-1, flowering and modifying the adaptation and grain yield devel- consistent with their previously described inhibitory roles in opment (Fig. 5). In other words, in wild-type soybeans, the EC (J flowering and transcriptional activation by E1 (64, 65). These interacts with heterodimers of LUX1-LUX2) has the strongest findings provide further evidence that soybean FT homologs suppressive effects on soybean flowering suppressors, and thus have diverged both in function and regulation. The divergence of promotes early flowering and low yield productivity. When a function among members of the FT/TERMINAL FLOWER 1 single mutation occurs in lux1 or lux2, J interacts with either (TFL1) gene family is well established in Arabidopsis. FT and homodimers of LUX1-LUX1 or LUX2-LUX2 to maintain the TFL1 encode a pair of flowering regulators with homology to AGRICULTURAL SCIENCES same suppressive activity as J-LUX1-LUX2 of EC without phosphatidylethanolamine-binding proteins; they share ∼60% phenotypic flowering changes as wild-types. However, the mu- amino acid sequence identity but function in an opposite manner tation of J reduced the suppressive activity of EC on the func- (66, 67). FT promotes the transition to flowering whereas TFL1 tions of E1 homologs, and thus resulted in late flowering and represses this transition (68, 69). high yield. Strikingly, the double mutant of lux1 lux2 completely As expected, the circadian clock genes, three orthologs of impaired the functions of EC, and thus fully released the func- Arabidopsis NIGHT LIGHT–INDUCIBLE AND CLOCK- tions of three E1 suppressors and resulted in extreme late- REGULATED 3 (LNK3), and four homologs of CYCLING flowering phenotypes (Fig. 5). DOF (CDF) were significantly induced in the double mutant. In

ABCD wild type single mutation mutation of j double mutaon lux1 lux2 of lux1 lux2 of or x

E1s E1s

or E1s J protein E1s

E1s LUX1 protein

LUX2 protein

extreme late flowering early flowering early flowering late flowering yield not low yield low yield high yield applicable

Fig. 5. Model summarizing the mechanism of SEC functions under SD conditions. J protein physically associates with LUX1 and LUX2 proteins in which LUX1 interacts with LUX2 to form SEC J-LUX1-LUX2 and directly bind to the promoters of E1 and its two homologs E1La and E1Lb to suppress their expressions, thus mediating the transcriptional suppression of FTs to control flowering and adaptation and grain-yield productivity. (A) In wild-type soybeans, the SEC (J interacts with heterodimers of LUX1-LUX2) has the strongest suppressive effects on soybean flowering suppressors thus promotes early flowering and low yield productivity. (B)In single mutant of either of lux1 or lux2, J interacts with either homodimers of LUX1-LUX1 or LUX2-LUX2 to maintain the same suppressive activity as J-LUX1-LUX2 of SEC without phenotypic flowering changes. (C) The mutation of J reduced the activity of SEC thus resulted in late flowering and high yield. (D) Double mutant of lux1 lux2 completely impairs the functions of SEC and thus fully releases the functions of three E1 suppressors resulting in extreme late-flowering phenotypes.

Bu et al. PNAS | 7of10 A critical role of the soybean evening complex in the control of photoperiod sensitivity and https://doi.org/10.1073/pnas.2010241118 adaptation Downloaded by guest on September 25, 2021 Arabidopsis, lux mutants show arrhythmic (15); therefore, we with a series of continuums of flowering time and adaptability. examined whether it is true in soybean. The free-running period These cultivars will be extremely important for the adaptation of leaf movement rhythmicity in W82, lux1, lux2-1, and lux1 lux2- and yield improvement in tropical countries to maximize the 1 plants under constant light (LL) was investigated. Unlike the soybean productivity. arrhythmicity of lux mutants in Arabidopsis, we found that the The roles of EC are well conserved in maintaining circadian period length in leaf movement of lux1 and lux2-1 single mutant rhythms and regulating flowering time in different plant species was ∼1.5 and 1 h longer than that in the wild-type, while the (14–26). The mutation of LUX leads to early-flowering pheno- lux1 lux2-1 double mutant displayed a longer period than either types in Arabidopsis and pea (15, 26). In addition, disruption of ∼ of the single mutants, having a period 6 h longer than that in LUX homologs has also been proposed as the molecular basis for wild-type plants (SI Appendix, Fig. S9 and Table S1). This indi- mutations conferring photoperiod-insensitive early flowering in cates that LUX1 and LUX2 function redundantly to maintain barley (Hordeum vulgare) early maturity10 (eam10) mutant (19) circadian rhythms in soybean, and a simultaneous mutation of and in einkorn wheat (Triticum monococcum) earliness per se 3 them could not completely abolish the rhythmicity, which is in- m (Eps-3A ) mutant (20). In contrast to these LD plant (LDP) consistent with Arabidopsis. In addition, the homologs of GA species, here we found that disruption of LUX1 and LUX2 delays pathway genes—including GA2oxidase 8, GIBBERELLIC ACID- STIMULATED ARABIDOPSIS 4 (GASA4), and GASA6—were flowering in soybean, a typical SDP species. This situation is similar with another EC component ELF3, which inhibits flow- up-regulated in the double-mutant plants, which may partially – explain the continuous growth of the mutant of Guangzhou ering in LDP species barley, wheat, pea, and lentil (18, 21 23). In Mammoth. We next performed qRT-PCR experiments to contrast, in the SDP species rice and soybean, ELF3 promotes verify the expression of several key genes that may participate flowering by suppressing expression of the key FT repressors in flowering identified in our RNA-seq analysis; the results Grain number, plant height and heading date 7 (Ghd7) and E1, indicated that the expression of these genes was consistent respectively (24, 25, 34, 72). This further supports the emerging with the RNA-seq results (SI Appendix,Fig.S8B). All of these view that upstream components of the photoperiod response results suggest that multiple gene pathways might be regulated pathway play opposite roles in SDP and LDP (18, 21–25). The by the EC in soybean to control flowering, maturity, and yield soybean J gene is the ortholog of Arabidopsis ELF3, and J pro- development. motes flowering through repressing the transcriptional expression of the legume-specific flowering repressor E1 under SD, Discussion which makes J a major source of adaptation and yield im- Precise flowering time is critical to crop adaptation and pro- provement in low-latitude regions (34). In LD species Arabi- ductivity in a given environment. Although the importance of dopsis and pea, mutants for the three EC components ELF3, flowering time in the adaptation and yield of soybean is well ELF4, and LUX have similar early-flowering phenotypes (14–18, established and several major genes have been characterized (32, 26), indicating that functions of EC genes are conserved in dif- 34), the molecular understanding of flowering is still insufficient. ferent species. Both ELF3 and ELF4 are single-copy genes in Our results indicate that the regulatory modules of soybean – diploid legumes, while there are three homologs of ELF3 (J, EC E1/E1 homologs are the key molecular networks controlling ELF3b-1, and ELF3b-2) and two homologs of ELF4 (ELF4a and soybean flowering and photoperiodism. The complete impair- ELF4b) in soybean (17, 18, 34). Like LUX1 and LUX2, the ho- ment of EC, which was reflected by the mutant of Guangzhou mologs of ELF3 and ELF4 might also play redundant roles in Mammoth, fully derepresses the transcriptions of three core soybean flowering repressors E1, E1La, and E1Lb, thus highly regulating flowering according to the dosage balance hypothesis. repressing FT homologs to extremely delay flowering even under Further genetic and molecular characterization of EC genes, inductive SD conditions. The Guangzhou Mammoth showed such as duplicated homologs of ELF4 and ELF3, are needed to extreme late flowering under NSD (13-h light/11-h dark) con- extend our understanding of the mechanisms of flowering and ditions in Guangzhou (23° 16′ N, 113° 23′ E), which flowered 11 adaptation in soybean. Exploration of the roles of EC in other wk later than wild-type (Fig. 4A). However, under ASD (12-h crop species will be of great important for the improvement of light/12-h dark, 25 °C) conditions in the growth chamber, the crop adaptation and grain yield. Guangzhou Mammoth flowered about 53 d later than wild-type In summary, we conclude that the two soybean LUX homo- (SI Appendix, Fig. S6). The flowering time has long been known logs, LUX1 and LUX2, like J, play essential roles in regulating to be regulated primarily by photoperiod and temperature (70, flowering and adaptation. Both LUX1 and LUX2 interact with J 71). The extremely late-flowering phenotypes of Guangzhou to form EC, and they are functionally redundant in promoting Mammoth under NSD conditions might be caused by the in- flowering. The EC represses E1s expression by binding to the teractive photo-thermal effects on soybean flowering. Moreover, LBS of their promoters, further relieving the expression of FT2a the flowering time of Guangzhou Mammoth under ALD and and FT5a and promoting flowering under SD conditions. Our ASD conditions showed no significant difference (SI Appendix, findings provide evidence of the critical functions of EC in Fig. S6), indicating that loss of function of EC leads to flowering-time control and latitudinal adaptation and yield devel- photoperiod-insensitivity of Guangzhou Mammoth. opment that will be greatly helpful in soybean molecular breeding. These results indicate that soybean EC plays an essential role in controlling flowering and photoperiod sensitivity, conse- Materials and Methods quently determining the adaptation and yield development. The The soybean [G. max (L.) Merr.] cultivar W82 was used as the wild-type. extreme late flowering of Guangzhou Mammoth further suggests Methodological details of plant growth, gene-expression analysis, nonlinear that the EC may integrate the photoreceptors and circadian regression analysis, phylogenetic analysis, protein–protein interaction as- clocks to control E1 expressions to mediate photoperiod flow- says, transient dual-luciferase assay, Western blot, protein–DNA interaction ering in soybean. Genetic dissections of the relationship between assays, transcriptome analysis, and leaf movement experiments are de- these components with EC will be helpful to understand the scribed in SI Appendix, Materials and Methods. The primers used in this molecular mechanisms of EC in soybean flowering. Therefore, study are listed in Dataset S3. this EC–E1 regulatory module provides us with a promising perspective to generate various allelic combinations of J, LUX1, Data Availability. RNA-seq data have been deposited in the Sequence Read LUX2, E1, E1La, and E1Lb by a CRISPR/Cas9 genome-editing Archive (NCBI-SRA) (BioProject no. PRJNA628851). All other study data are approach in the elite cultivars to quickly develop new cultivars included in the article and supporting information.

8of10 | PNAS Bu et al. https://doi.org/10.1073/pnas.2010241118 A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. This work was supported by National Natural Science Lu), and 31930083 (to B.L.). This work was also funded by the Major Program of Foundation of China Grants 31725021 (to F.K.), 31901500 (to T.B.), 31701445 (to S. Guangdong Basic and Applied Research Grant 2019B030302006 (to F.K. and B.L.).

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10 of 10 | PNAS Bu et al. https://doi.org/10.1073/pnas.2010241118 A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation Downloaded by guest on September 25, 2021