The HD-Zip transcription factor SlHB15A regulates abscission by modulating jasmonoyl-isoleucine biosynthesis

Abstract

Plant organ abscission, a process that is important for development and reproductive success, is inhibited by the phytohormone auxin and promoted by another phytohormone, jasmonic acid (JA). However, the molecular mechanisms underlying the antagonistic effects of auxin and JA in organ abscission are unknown. We identified a tomato (Solanum lycopersicum) class III homeodomain-leucine zipper transcription factor, HOMEOBOX15A (SlHB15A), which was highly expressed in the flower pedicel abscission zone and induced by auxin. Knocking out SlHB15A using clustered regularly interspaced short palindromic repeats-associated protein 9 technology significantly accelerated abscission. In contrast, overexpression of microRNA166-resistant SlHB15A (mSlHB15A) delayed abscission. RNA sequencing and reverse transcription-quantitative PCR analyses showed that knocking out SlHB15A altered the expression of genes related to JA biosynthesis and signaling. Furthermore, functional analysis indicated that SlHB15A regulates abscission by depressing JA-isoleucine (JA-Ile) levels through inhabiting the expression of JASMONATE-RESISTANT1 (SlJAR1), a gene involved in JA-Ile biosynthesis, which could induce abscission-dependent and abscission-independent ethylene signaling. SlHB15A bound directly to the SlJAR1 promoter to silence SlJAR1, thus delaying abscission. We also found that flower removal enhanced JA-Ile content and that application of JA-Ile severely impaired the inhibitory effects of auxin on abscission. These results indicated that SlHB15A mediates the antagonistic effect of auxin and JA-Ile during tomato pedicel abscission, while auxin inhibits abscission through the SlHB15A-SlJAR1 module.

Figure 1

RT-qPCR and in situ hybridization of abscission-related SlHB15A and SlHB15B expression. A, Relationship between Arabidopsis and tomato HD-ZIP III proteins. The phylogenetic analysis was performed using MEGA version 6 software. Detailed information regarding the tomato sequences is listed in Supplemental Table S3. B and C, RT-qPCR analysis of SlHB15A (B) and SlHB15B (C) expression in the AZs, as well as, proximal and distal tissues of tomato pedicels. The results represent mean of three biological replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05). D–F, In situ hybridization of SlHB15A and SlHB15B in tomato pedicel explants. The 3′-labeled SlHB15A sense probes were used as negative controls (D). WT pedicel sections were hybridized with 3′-labeled antisense (E) SlHB15A and (F) SlHB15B probes. Dis, distal; Pro, proximal. Scale bar: 100 µm. G and H, RT-qPCR analysis of SlHB15A (G) and SlHB15B (H) expression in tomato pedicel AZs from the control (tomato pedicel with flower), flower removal (FR), flower removal with auxin treatment, and flower removal with aspirin treatment groups. Values represent mean of three biological replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05). I, RT-qPCR analysis of SlHB15A and SlHB15B expression in the tomato pedicel AZs under 1-MCP treatment. Values represent mean of three biological replicates. Asterisks indicate significantly different values (Student’s t test, **P < 0.01 and *P < 0.05).

Figure 2

Pedicel abscission assay in WT plants as well as the 35S:mSlHB15A, CR-slhb15a, CR-slhb15b, and CR-slhb15aslhb15b lines. A, The effect of flower removal on WT, 35S:mSlHB15A (35S #3, 35S #5, and 35S #6), and CR-slhb15a (CR-slhb15a #1, CR-slhb15a #5, and CR-slhb15a #7) explant pedicel abscission. The results represent mean of three replicates ± sd, with at least 15 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05). B, The effect of flower removal on WT, CR-slhb15a, CR-slhb15b, and CR-slhb15aslhb15b explant pedicel abscission. The results represent mean of three replicates ± sd, with at least 15 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05).

Figure 3

The KEGG classification of assembled DEGs and variation in expression of JA-related genes in the AZs of CR-slhb15a and 35S:mSlHB15A lines, when compared with those of WT plants. A, KEGG classification of assembled DEGs. The most represented pathway was hormone signal transduction (15 DEGs). Two biological replicates were used in this RNA-seq experiment. B, RT-qPCR analysis of SlAFP3, SlMYC2-like, SlJAR1, SlGH3.10, cytochrome P450 (CYP)94B3, and CYP 94C1 in the AZs from WT plants, CR-slhb15a lines, and 35S:mSlHB15A lines. The results represent mean of three replicates ± sd, with at least 20 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05).

Figure 4

JA and JA-Ile concentrations in pedicel AZs. A, Quantification of JA levels in the AZs of WT, CR-slhb15a, and 35S:mSlHB15A lines during abscission, using LC-MS/MS. The results represent mean of three replicates ± sd, with at least 20 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05). B, Quantification of JA-Ile in AZs of WT, CR-slhb15a, and 35S:mSlHB15A lines during abscission, using LC-MS/MS. The results represent mean of three replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05).

Figure 5

Effects of silencing SlJAR1 and SlGH3.10 on the kinetics of tomato pedicel abscission and JA-Ile content. A, Kinetics of pedicel abscission in TRV, TRV-SlJAR1, and TRV-SlGH3.10 lines. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent mean of three replicates ± sd, with at least 15 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05). B, Quantification of JA-Ile in the AZs of TRV, TRV-SlJAR1, and TRV-SlGH3.10 lines during abscission, using LC-MS/MS. The results represent mean of three replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05). C, The effect of JA-Ile on abscission of TRV and TRV-SlJAR1 lines. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent mean of three replicates ± sd, with at least 15 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05).

Figure 6

SlJAR1-dependent JA-Ile-induced abscission is ethylene dependent as well as independent. A, RT-qPCR analysis of SlJAR1 expression in the AZs of WT lines treated with or without the ethylene inhibitor, 1-MCP. The results represent mean of three replicates ± sd, with at least 20 samples per replicate (Student’s t test, P < 0.05). B, The effect of JA-Ile on abscission in 1-MCP-treated WT plants. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent means of six biological replicates, with at least 15 samples per replicate. Upmost/lowest lines indicate the maximum/minimum values; Box limits indicate the upper and lower quartiles; Lines in boxes indicate the median values of these data. Open squares represent the mean value and filled circles represent each data points. Asterisks indicate significantly different values (Student’s t test, *P < 0.05). C, The effect of JA-Ile on ethylene production in WT AZ explants. Three independent experiments were performed. Data have been expressed as mean ± sd (Student’s t test, *P < 0.05 and **P < 0.01). D, Kinetics of pedicel abscission in TRV and TRV-SlJAR1 lines, after 1-MCP treatment. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent mean of six replicates, with at least 15 samples per replicate. Upmost/lowest lines indicate the maximum/minimum values; Box limits indicate the upper and lower quartiles; Lines in boxes indicate the median values of these data. Open squares represent the Figure 6 (continued) mean value and filled circles represent each data points. Asterisks indicate significantly different values (Student’s t test, **P < 0.01). E, Kinetics of pedicel abscission in TRV-SlEIN2 lines, after JA-Ile treatment. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent mean of six replicates, with at least 15 samples per replicate. Upmost/lowest lines indicate the maximum/minimum values; Box limits indicate the upper and lower quartiles; Lines in boxes indicate the median values of these data. Open squares represent the mean value and filled circles represent each data points. Asterisks indicate significantly different values (Student’s t test, **P < 0.01).

Figure 7

SlHB15A represses SlJAR1 expression by binding to its promoter. A, The interaction between SlHB15A and the SlJAR1 promoter ATGAT sequence was assessed using Y1H assays. Co-transformed AD-Rec-P53 and P53-promoter fragments in Y1HGold were used as positive controls. Empty vector and SlJAR1 promoter fragments were used as negative controls. B, The interaction between SlHB15A and the SlJAR1 promoter ATGAT sequence was assessed using EMSA. The hot probe was a biotin-labeled SlJAR1 promoter fragment containing the ATGAT element, while the cold probe was a nonlabeled competitive probe (at a 200-fold concentration of the hot probe). The mutant cold probe was the unlabeled hot probe sequence with two mutated nucleotides. The bands and free probes have been annotated using arrowheads. C, ChIP-qPCR analysis of direct SlHB15A binding to the SlJAR1 promoter. Cross-linked chromatin samples were extracted from mSlHB15A-GFP overexpressing AZs and precipitated using an anti-GFP antibody. Eluted DNA was used to amplify the sequences neighboring the ATGAT element, using qPCR. The 35S:GFP line was used as negative control. Values represent mean of three replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05).

Figure 8

The SlHB15A-SlJAR1 module is involved in tomato pedicel abscission. A, The effect of JA-Ile on abscission in the WT and 35S:mSlHB15A lines. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent mean of three replicates ± sd, with at least 12 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05). B, Abscission assay in CR-slhb15a-TRV and CR-slhb15a-TRV-SlJAR1 lines. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent mean of three replicates ± sd, with at least 12 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05). C, Abscission assay in CR-slhb15a plants treated with or without the JA-Ile inhibitor, jarin-1. The percentages of pedicel abscission were determined at intervals after flower removal. The results represent mean of three replicates ± sd, with at least 12 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05). D, Quantification of JA-Ile in the AZs of WT plants treated with or without auxin during abscission, using LC-MS/MS. The results represent mean of three replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05). E, RT-qPCR analysis of SlJAR1 expression in WT AZs treated with or without auxin. The results represent mean of three replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05). F, Auxin inhibition abscission assay in the WT, 35S:mSlHB15A, and CR-slhb15a lines. The percentages of pedicel abscission were determined at intervals after flower removal and the pedicels were incubated with 30 µg·L⁻¹ auxin. Values represent mean of three replicates ± sd, with 15 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05). G, RT-qPCR analysis of SlJAR1 expression in AZs of WT, 35S: mSlHB15A, and CR-slhb15a plants treated with auxin. The results represent mean of three replicates ± sd. Different letters indicate significant differences (Student’s t test, P < 0.05). H, Abscission assay in auxin-treated explants treated with or without JA-Ile. The percentages of pedicel abscission were determined at intervals after flower removal. Values represent mean of three replicates ± sd, with 15 samples per replicate. Different letters indicate significant differences (Student’s t test, P < 0.05).

Figure 9

Model showing SlHB15A-mediated repression of SlJAR1 expression by auxin, which prevents JA-Ile accumulation, resulting in inhibition of abscission. Under a normal environment (left panel), auxin promotes SlHB15A expression to inhibit SlJAR1 expression and prevent abscission. After auxin depletion (right panel), miR166 expression increases and SlHB15A expression decreases, following which SlJAR1 is induced to synthesize JA-Ile, which accelerates abscission. The arrows represent positive regulation and the bars represent inhibition. miR166, micro-RNA 166; HB15A, homeobox 15A; JAR1, jasmonate-resistant 1; ETR, ethylene receptor; CTR1, constitutive triple response 1; EIN2, ethylene insensitive 2.