A SlCLV3-SlWUS module regulates auxin and ethylene homeostasis in low light-induced tomato flower abscission

Abstract

Premature abscission of flowers and fruits triggered by low light stress can severely reduce crop yields. However, the underlying molecular mechanism of this organ abscission is not fully understood. Here, we show that a gene (SlCLV3) encoding CLAVATA3 (CLV3), a peptide hormone that regulates stem cell fate in meristems, is highly expressed in the pedicel abscission zone (AZ) in response to low light in tomato (Solanum lycopersicum). SlCLV3 knockdown and knockout lines exhibit delayed low light-induced flower drop. The receptor kinases SlCLV1 and BARELY ANY MERISTEM1 function in the SlCLV3 peptide-induced low light response in the AZ to decrease expression of the transcription factor gene WUSCHEL (SlWUS). DNA affinity purification sequencing identified the transcription factor genes KNOX-LIKE HOMEDOMAIN PROTEIN1 (SlKD1) and FRUITFULL2 (SlFUL2) as SlWUS target genes. Our data reveal that low light reduces SlWUS expression, resulting in higher SlKD1 and SlFUL2 expression in the AZ, thereby perturbing the auxin response gradient and causing increased ethylene production, eventually leading to the initiation of abscission. These results demonstrate that the SlCLV3-SlWUS signaling pathway plays a central role in low light-induced abscission by affecting auxin and ethylene homeostasis.

Figure 1

Flower pedicel abscission is enhanced under low-light conditions in a SlCLV3-dependent manner. A, RT-qPCR analysis of relative SlCLV3 expression levels in the AZ after 0, 1, 3, 5, and 7 days under normal light or low-light conditions. Data are means ± standard deviation (SD) of three independent pools of AZ from different plants. Significant differences were determined by two-way ANOVA with Sidak’s test: ^*P < 0.05; ^****P < 0.0001. B, Representative GUS staining pattern of CLV3pro:GUS inflorescences showing the expression of SlCLV3 in the AZ after growth under control and 70% shading conditions for 7 days. Three independent transgenic lines were stained for each condition. Scale bars, 1 cm. C, Representative inflorescence phenotype of the WT (tomato Ailsa Craig) and TAPG4pro:SlCLV3-RNAi lines 11, 17, and 33 under low-light conditions. Arrows indicate abscised flowers. Scale bar, 1 cm. D, Frequency of flower abscission in the WT and TAPG4pro:SlCLV3-RNAi lines 11, 17, and 33 under low-light conditions. Flower abscission was scored until fruit set. Five plants were scored for each genotype. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^****P < 0.0001. E, Frequency of flower abscission in the WT and the mutants Slclv3-8, Slclv3-13, and Slclv3-15 under low-light conditions. Flower abscission was scored until fruit set. Five plants were scored for each genotype. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^****P < 0.0001. F, Time course of pedicel abscission for WT, Slclv3, and TAPG4pro:SlCLV3-RNAi plants treated with H₂O, sSlCLV3, or SlCLV3p peptide. Data are means ± SD of six independent treatments, with at least 10 pedicels per treatment. Different letters represent significant differences, as determined by two-way ANOVA with Tukey’s test, P < 0.05.

Figure 2

SlCLV3 acts primarily through the receptor kinases SlCLV1 and SlBAM1. A, RT-qPCR analysis of SlCLV1, SlCLV2, SlCRN, SlBAM1, SlBAM2, SlBAM3, and SlBAM4 relative expression levels in the AZ. Data are means ± SD of three independent pools of AZ from different plants. Significant differences were determined by two-way ANOVA with Sidak’s test; ^****P < 0.0001. B, Frequency of flower abscission in WT, Slclv1, Slbam1, and Slclv1 Slbam1 plants under low-light conditions. Flower abscission was scored until fruit set. Two plants were scored for each of the three independent plant lines. Significant differences were determined by one-way ANOVA with Tukey’s test; ^*P < 0.05; ^****P < 0.0001. C, Time course of pedicel abscission for the WT, Slclv1, Slbam1, and Slclv1 Slbam1 plants treated with sSlCLV3 or SlCLV3p peptide. Data are means ± SD of six independent treatments, with at least 10 pedicels per treatment. Different lowercase letters represent significant differences, as determined by one-way ANOVA with Duncan’s test; P < 0.05.

Figure 3

SlCLE2 compensates for the loss of SlCLV3 for abscission. A, RT-qPCR analysis of SlCLE2 relative expression levels in the AZ of WT, Slclv3-8, and Slclv3-15 plants. Data are means ± SD of three independent pools of the AZ. Significant differences were determined by two-way ANOVA with Dunnett’s test compared to the WT; ^*P < 0.05; ^***P < 0.0005. B, RT-qPCR analysis of SlCLE2 relative expression levels in the AZ of WT, Slclv3, and TAPG4pro:SlCLV3-RNAi plants under normal and low-light conditions. Data are means ± SD of three independent pools of AZ from different plant lines. Significant differences were determined by two-way ANOVA with Sidak’s test compared to the WT grown in normal-light conditions (Light); ^*P < 0.05; ^**P < 0.01; ^***P < 0.0005. C, Frequency of flower abscission in WT, Slclv3, Slcle2, and Slclv3 Slcle2 under low-light conditions. Flower abscission was scored until fruit set. Three biologically independent plant lines were treated. Two biologically independent treatments were performed for each plant line. Significant differences were determined by one-way ANOVA with Tukey’s test; ^***P < 0.0005; ^****P < 0.0001. D, Time course of WT flower pedicel explant abscission rates after treatment with different versions of SlCLV3 or SlCLE2 peptide at 8, 16, 24, 32, and 40 h after flower removal. SlCLV3p and SlCLE2p, triarabinosylated peptides. Control, water. Data are means ± SD of six independent treatments, with at least 10 pedicels per treatment. Different letters indicate significant differences, as determined by two-way ANOVA with Tukey’s test; P < 0.05. E, Time course of pedicel abscission for WT, Slclv1, Slbam1, and Slclv1 Slbam1 plants after treatment with sSlCLE2 or SlCLE2p peptide. Data are means ± SD of six independent treatments, with at least 10 pedicels per treatment. Different letters indicate significant differences, as determined by one-way ANOVA with Duncan’s test; P < 0.05.

Figure 4

SlCLV3 regulates abscission by repressing SlWUS expression. A, Relative expression levels of SlWUS in the AZ of Slclv3, Slclv1, Slbam1, Slclv3 Slcle2, and Slclv1 Slbam1 plants. Data are means ± SD of three independent pools of AZ. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^****P < 0.0001. B and C, Time course of pedicel abscission for the WT, SlWUS-RNAi (lines 5, 7, and 8) (B), and SlWUS-OX lines (62, 65, and 68) (C). Each point is the mean ± SD of three biologically independent tests in each plant line, with at least 10 pedicels per test. Significant differences were determined by two-way ANOVA with Dunnett’s test; ^*P < 0.05; ^**P < 0.01; _****P < 0.0001. D, Frequency of flower drop in WT, SlWUS-OX (lines 62, 65, and 68), and SlWUS-RNAi (lines 5, 7, and 8) plants under low-light conditions. Flower abscission was scored until fruit set. Six plants were scored for each plant line. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^****P < 0.0001.

Figure 5

SlWUS directly represses SlKD1 and SlFUL2 expression in the tomato AZ. A, RT-qPCR analysis of the relative expression levels of auxin- and ethylene-related transcription factor genes in the AZ of WT, SlWUS-RNAi, and SlWUS-OX plants. Data are means ± SD of three independent pools of AZ from different plant lines. Significant differences were determined by two-way ANOVA with Dunnett’s test compared to the WT; ^*P < 0.05; ^**P < 0.01; ^***P < 0.0005. B, RT-qPCR analysis of SlKD1 and SlFUL2 expression levels in the AZ of WT, Slclv3 Slcle2, and Slclv1 Slbam1 plants. Data are means ± SD of three independent pools of AZ from different plant lines. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^****P < 0.0001. C, Genome browser view of the distribution of DAP-seq reads (blue, input; red, WUS; pink, peak location) over the SlKD1 and SlFUL2 genomic regions. D, Yeast one-hybrid assay showing that SlWUS directly binds to the SlKD1 and SlFUL2 promoters. The combination of AD-Rec-P53 and P53-Promoter was used as the positive control, while AD-Empty and KD1-Promoter and FUL2-Promoter were the negative control. Aureobasidin A treatment (200 ng/L) is indicated at the bottom. E, Schematic diagram of the SlKD1 and SlFUL2 promoter fragments containing the binding sequence used for the Y1H assay and EMSA. The gel mobility shift assay revealed direct binding of SlWUS to the “GAATGATT” sequence in the SlKD1 promoter and “TTCATTCA” in the SlFUL2 promoter. The probe sequence from the SlKD1 and SlFUL2 promoters is shown, with red letters representing the binding motif. WT, probe with intact sequence. A mutated probe and a 200-fold excess amount of unlabeled probe were added as competitors to the binding reaction. The retarded bands and the free probes are indicated by arrowheads. F, LUC activity measured after transient infiltration of SlKD1pro:LUC or SlFUL2pro:LUC and 35S:SlWUS constructs in N. benthamiana leaves. Data are means ± SD of three biologically independent treatments. Significant differences were determined by one-way ANOVA with Dunnett’s test; ^****P < 0.0001).

Figure 6

SlFUL2 positively regulates flower pedicel abscission. A, RT-qPCR analysis of SlFUL1 and SlFUL2 relative expression levels at 0, 2, 4, 8, and 16 h after flower removal in the AZ. Data are means ± SD of three independent pools of AZ from different plants. B, Time course of pedicel abscission in the WT, SlFUL1/SlFUL2-RNAi, and SlFUL2-OX plants at 8 and 16 h after flower removal. Data are means ± SD of three independent tests in each plant line, with at least 10 pedicels per treatment. Significant differences were determined by two-way ANOVA with Dunnett’s test compared to the WT; ^*P < 0.05; ^**P < 0.01; ^***P < 0.0005; ^****P < 0.0001). C, Ethylene production in the AZ of WT, SlFUL1/SlFUL2-RNAi, and SlFUL2-OX plants, analyzed 8 h after flower removal. Data are means ± SD of three independent tests in each plant line. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^*P < 0.05; ^**P < 0.01.

Figure 7

SlKD1 and SlFUL2 act downstream of the low light-induced SlCLV3 signaling pathway. A, Expression pattern of DR5:GUS in the AZ of WT, Slclv3 Slcle2, Slclv1 Slbam1, and SlWUS-RNAi plants. Transverse sections were made from the distal region, the distal side of the AZ, the proximal side of the AZ, and the proximal region. Three independent plant lines were observed, with at least 10 pedicels per plant line. B and C, Ethylene production in the AZ of WT, Slclv3 Slcle2 (B), Slclv1 lbam1 (B), SlWUS-RNAi (C), and SlWUS-OX (C) plants, analyzed 8 h after flower removal. Data are means ± SD of three independent tests, with at least 10 pedicels from different plant lines per test. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^***P < 0.0005. D and E, RT-qPCR analysis of SlKD1 (D) and SlFUL2 (E) expression levels in the AZ under normal light and shading conditions. Data are means ± SD of three independent pools of AZ from different plants. Significant differences were determined by Student’s t test; ^*P < 0.05; ^**P < 0.01. F, Frequency of flower abscission in WT, SlFUL1/SlFUL2-RNAi, SlKD1-RNAi, and SlFUL1/SlFUL2-RNAi SlKD1-RNAi plants under low-light conditions. Flower abscission was scored until fruit set. Six plants were scored for each genotype. Significant differences were determined by one-way ANOVA with Tukey’s test; ^*P < 0.05; ^****P < 0.0001. G, Number of days to reach 50% abscised flowers in WT, Slclv3 Slcle2, Slclv3 Slcle2 SlFUL2-OX, Slclv3 Slcle2 SlKD1-OX, and Slclv3 Slcle2 SlFUL2-OX SlKD1-OX plants. Six independent tests were scored, with at least 10 pedicels per test. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^*P < 0.05; ^****P < 0.0001. H, Number of days to reach 50% abscised flowers in WT, SlWUS-OX, SlWUS-OX SlFUL2-OX, SlWUS-OX SlKD1-OX, and SlWUS-OX SlFUL2-OX SlKD1-OX plants. Six independent tests were scored, with at least 10 pedicels per test. Significant differences were determined by one-way ANOVA with Dunnett’s test compared to the WT; ^*P < 0.05; ^****P < 0.0001.

Figure 8

Model of low light-induced modulation of tomato flower pedicel abscission by SlCLV3- SlWUS. Low light stimulates the accumulation of SlCLV3 in the AZ, which is perceived by SlCLV1 and SlBAM1. After a series of signal transmissions, SlWUS expression is repressed in the AZ. In the absence of SlCLV3 (as in Slclv3 mutants), SlCLE2 can compensate for its function in regulating abscission. SlWUS acts as a negative regulator of abscission. Upon activation of the SlCLV3-SlWUS signaling pathway, the expression of SlKD1 and SlFUL2 is induced, the auxin response gradient in the AZ is disturbed, and ethylene production increases, leading to abscission.