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

Abstract

Photoperiod sensitivity is a key factor in plant adaptation and crop production. In the short-day plant soybean, adaptation to low latitude environments is provided by mutations at the J locus, which confer extended flowering phase and thereby improve yield. The identity of J as an ortholog of Arabidopsis ELF3, a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles. Here we show that the two soybean homeologs of LUX ARRYTHMO interact with J to form a soybean EC. Characterization of mutants reveals that these genes are highly redundant in function but together are critical for flowering under short day, where the lux1 lux2 double mutant shows extremely late flowering and a massively extended flowering phase. This phenotype exceeds that of any soybean flowering mutant reported to date, and is strongly reminiscent of the "Maryland Mammoth" tobacco mutant that featured in the seminal 1920 study of plant photoperiodism by Garner and Allard [W. W. Garner, H. A. Allard, J. Agric. Res. 18, 553-606 (1920)]. We further demonstrate that the J-LUX complex suppresses transcription of the key flowering repressor E1 and its two homologs via LUX binding sites in their promoters. These results indicate that the EC-E1 interaction has a central role in soybean photoperiod sensitivity, a phenomenon also first described by Garner and Allard. EC and E1 family genes may therefore constitute key targets for customized breeding of soybean varieties with precise flowering time adaptation, either by introgression of natural variation or generation of new mutants by gene editing.

Keywords: LUX ARRHYTHMO (LUX); adaptation; evening complex (EC); flowering; soybean.

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.

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.

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. In C, Guangzhou Mammoth is 210-cm high and in D, it grows up to 250-cm high without stopping growing.

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) in W82, 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.

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.