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Mobile TERMINAL FLOWER1 determines seed size in Arabidopsis

Nature Plants volume 6, pages 1146–1157 (2020)Cite this article

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Abstract

Seed size is a pivotal agronomic trait that links plant sexual reproduction and subsequent seedling establishment, and is affected by the timing of endosperm cellularization following endosperm proliferation after double fertilization. The molecular switch that controls the timing of endosperm cellularization has so far been largely unclear. Here, we report that the Arabidopsis TERMINAL FLOWER1 (TFL1) is a mobile regulator generated in the chalazal endosperm, and moves to the syncytial peripheral endosperm to mediate timely endosperm cellularization and seed size through stabilizing ABSCISIC ACID INSENSITIVE 5. We further show that Ras-related nuclear GTPases interact with TFL1 and regulate its trafficking to the syncytial peripheral endosperm. Our findings reveal TFL1 as an essential molecular switch for regulating endosperm cellularization and seed size. Generation of mobile TFL1 in the chalazal endosperm, which is close to maternal vascular tissues, could provide a hitherto-unknown means to control seed development by mother plants.

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Fig. 1: TFL1 affects seed size.
Fig. 2: Endosperm cellularization is delayed in tfl1-20.
Fig. 3: TFL1 mRNA and protein localization in developing seeds.
Fig. 4: TFL1 interacts with RAN proteins.
Fig. 5: Dominant-negative RAN2 (DN-ran2) phenocopies loss of function of TFL1 in seed development.
Fig. 6: TFL1 regulates seed size partially through affecting ABI5 stability.

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Data availability

We declare that all data supporting the findings of this study are available within the article and its supplementary information files or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the Arabidopsis Biological Resource Centre for providing various mutants, and the members of the Yu laboratory for discussion and comments on the manuscript. This work was supported by the Singapore National Research Foundation Investigatorship Programme (NRFNRFI2016-02), the Agency for Science, Technology and Research (A*STAR) under its Industry Alignment Fund—Pre-Positioning (IAF-PP) (A19D9a0096), and intramural research support from the National University of Singapore and Temasek Life Sciences Laboratory.

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Authors and Affiliations

  1. Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore

    Bin Zhang, Chengxiang Li, Yan Li & Hao Yu

  2. Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore

    Bin Zhang, Chengxiang Li & Hao Yu

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  1. Bin Zhang
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Contributions

B.Z. and H.Y. conceived and designed the project. B.Z., C.L. and Y.L. performed the experiments. B.Z. and H.Y. analysed the data and wrote the paper.

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Correspondence to Hao Yu.

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Peer review information Nature Plants thanks Steven Penfield and Xian Sheng Zhang for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 gTFL1-3HA and gTFL1-GFP rescue the early flowering and terminal flower phenotypes of tfl1-20.

a, tfl1-20 flowers much earlier than a wild-type (WT) plant and independent tfl1-20 gTFL1-3HA or tfl1-20 gTFL1-GFP transgenic lines. b, Independent tfl1-20 gTFL1-3HA and tfl1-20 gTFL1-GFP transgenic lines develop normal inflorescence apices like a WT plant, whereas tfl1-20 develops terminal flowers (a). Scale bars, 2 cm.

Extended Data Fig. 2 TFL1 affects endosperm cellularization.

a, Differential interference contrast (DIC) microscopy of cleared whole-mount seeds of wild-type (WT) and tfl1-20 plants at 1, 2, and 6 days after pollination (DAP). Scale bars, 50 µm. The experiment was repeated three times independently with similar results. b, Percentage of seeds with syncytial or cellularized peripheral endosperm in WT and tfl1-20 at 3 to 5 DAP examined by confocal microscopy. Randomly selected seeds from more than 10 siliques for each genotype were examined at each time point.

Extended Data Fig. 3 TFL1 mRNA and protein expression in Arabidopsis.

a, Quantitative real-time PCR analysis of TFL1 mRNA expression in various tissues (upper panel) or developing siliques at 2 to 7 days after pollination (DAP; lower panel) in Arabidopsis. OF, open flower; IS, inflorescence stem; RL, rosette leaf; Rt, root; Sil, silique; CL, cauline leaf; FB, flower bud. Results were normalized against the expression levels of the U-BOX gene as an internal control. Expression levels in the lower panel are shown as relative values to the 2 DAP level set as 1. Values are mean ± SD of three biological replicates. b, In situ localization of TFL1 expression in developing seeds at 4 DAP using the TFL1 antisense or sense probe. SPE, syncytial peripheral endosperm; CZE, chalazal endosperm. Scale bars, 100 μm. c, d, Localization of TFL1-GFP in an inflorescence meristem (c) and a root (d) of tfl1-20 gTFL1-GFP. Merge, merge of GFP and bright field images. Scale bars, 20 µm. e, Localization of TFL1-GFP in developing tfl1-20 gTFL1-GFP seeds at 4 DAP by cryosectioning under enhanced fluorescence intensity compared to those shown in Fig. 3d. TFL1-GFP signal is much weaker in the chalazal endosperm than in the syncytial peripheral endosperm (upper panel). The lower panel is a close-up view of the chalazal endosperm and its nearby syncytial peripheral endosperm shown above. BF, bright field image. CZE, chalazal endosperm; EM, embryo; Merge, merge of GFP and BF images; SPE, syncytial peripheral endosperm. Scale bars, 50 µm. f, Detection of background green fluorescence signal in a wild-type seed at 4 DAP. This image, acquired under the same condition for Fig. 3d, serves as a negative control for Fig. 3d. The auto-fluorescence (green fluorescence) signal was slightly detectable. Scale bar, 50 µm. g, Western blot analysis of TFL1-3HA protein (arrowhead) in selected tfl1-20 gTFL1-3HA transgenic lines that possibly contain one T-DNA insertion site based on their segregation ratios. Total protein was extracted from young siliques, and subjected to Western blot analysis using anti-HA antibody. The experiments in b–g were repeated three times independently with similar results.

Source data

Extended Data Fig. 4 Cytoplasmic localization of TFL1 in Arabidopsis.

a,b, Localization of TFL1-GFP in developing tfl1-20 gTFL1-GFP seeds stained with DAPI at 4 (a) and 5 (b) days after pollination (DAP) by cryosectioning. TFL1-GFP signal in the syncytial (a) and cellularized (b) peripheral endosperm does not co-localize with DAPI staining. The inset in each panel in (a) or (b) shows one enlarged nucleus of the syncytial or cellularized peripheral endosperm, respectively. DAPI, fluorescence of 4,6-diamino-2-phenylindole; BF, bright-field image; Merge, merge of GFP, DAPI and BF images. Scale bars, 50 µm. c, Analysis of TFL1-3HA cytoplasmic localization by immunogold electron microscopy using anti-HA antibody in the syncytial peripheral endosperm of tfl1-20 gTFL1-3HA seeds at 4 DAP. The lower panel shows a higher magnification of the area within the box indicated in the upper panel. Arrows indicate gold particles. There are no detectable gold particles in the nucleus. CV, central vacuole; iCW, cell wall of ii1; ii1, the innermost cell layer of the inner integument (endothelium cell); N, nucleus; Nu, nucleolus; PSV, protein storage vacuoles; SPE, syncytial peripheral endosperm. Scale bars, 2 µm. d, Subcellular localization of TFL1-GFP in the cells of a tfl1-20 gTFL1-GFP inflorescence meristem. Merge, merge of GFP and bright field images. Scale bar, 10 μm. The experiments in a-d were repeated three times independently with similar results.

Extended Data Fig. 5 Isolation of RAN1, RAN2, and RAN3 loss-of-function mutants.

a, Schematic diagrams showing RAN1, RAN2, and RAN3 genomic regions, and the CRISPR-Cas9 target sites (ran1-3 and ran1-4) or T-DNA insertion sites (ran2-1, ran3-1 and ran3-2). b, Quantitative analysis of RAN2 or RAN3 expression in their corresponding T-DNA insertion mutants. Results were normalized against the expression levels of the U-BOX gene as an internal control. Values are mean ± SD of three biological replicates. c,d, CRISPR/Cas9-mediated target mutagenesis of RAN1 in the wild-type (c) and ran2-1 (d) background. The CRISPR/Cas9 target sites in RAN1 are underlined in the upper panels. The newly created ran1-3 single mutant (c) and ran1-4 ran2-1 double mutant (d) contain a short deletion at the second exon and 1-bp thymine (T) insertion at the beginning of the fifth exon in RAN1 (both highlighted by the black frame), respectively. The positions of the mutations in ran1-3 and ran1-4 ran2-1 in RAN1 protein are indicated. e, Quantitative analysis of seed size parameters of ran1-3, ran2-1 and ran3-1. Box plots show medians (lines), interquartile ranges (boxes), and whiskers (extending 1.5 times the interquartile ranges) of seed size parameters (area, perimeter, length, and width) of seeds of different genotypes (WT, n = 692; ran1-3, n = 153; ran2-1, n = 516; ran3-1, n = 461). Value represents the percentage change (%) in a seed parameter of a mutant relative to the mean value of WT plants set as 100%. Asterisks indicate significant differences between WT plants and other genotypes (two-tailed Mann-Whitney test, P < 0.0001). n.s, no statistical difference. P values for WT versus ran1-3, ran2-1, and ran3-1 are 0.000193, < 1 × 10−15, and 0.461, respectively, for seed area; 0.0463, 4.378 × 10−7, and 0.988, respectively, for seed perimeter; 0.0333, 0.800, and 2.43 × 10−6, respectively, for seed length; and 0.000409, < 1 × 10−15, and 0.000164, respectively, for seed width.

Source data

Extended Data Fig. 6 Analysis of RAN1, RAN2 and RAN3 expression patterns.

a, Quantitative analysis of RAN1, RAN2 and RAN3 expression in various tissues (upper panels) or developing siliques at 2 to 7 days after pollination (DAP, lower panels). OF, open flower; IS, inflorescence stem; RL, rosette leaf; Rt, root; Sil, silique; CL, cauline leaf; FB, flower bud. Results were normalized against the expression levels of the U-BOX gene as an internal control. Expression levels in lower panels are shown as relative values to the 2 DAP level set as 1. Values are mean ± SD of three biological replicates. b, GUS staining of GUS-gRAN2 seeds at 1 to 5 DAP. Scale bars, 50 µm. The experiment was repeated three times independently with similar results.

Extended Data Fig. 7 Pull-down assay of the interaction between TFL1 and the dominant-negative (DN-ran2) or truncated versions of RAN2.

a, Schematic diagrams of DN-ran2 and RAN2 truncated (Del1-Del3) proteins that were fused to MBP. The full-length RAN2 protein contains the effector binding domain and acidic C-terminal domain implicated in protein-protein interaction. The DN-ran2 protein contains a mutation of threonine (T) at the residue 27 to asparagine (N), which is located near the effector-binding domain of RAN2. b, Pull-down assay result. MBP and various MBP fusion proteins were used as baits, and the corresponding loading control was stained with Ponceau S (lower panel). The input of the prey protein TFL1-4HA extracted from 35 S:TFL1-4HA siliques and its corresponding pull-downed signals were examined by immunoblot analysis using anti-HA antibody (upper panel). The experiment was repeated three times independently with similar results.

Source data

Extended Data Fig. 8 Seed size phenotypes of gDN-ran2-3FLAG (gDN) and TFL1:3FLAG-DN-ran2 (TFL1:DN) transgenic plants.

a,b, Western blot analysis of DN-ran2 protein expression in gDN (a) or TFL1:DN (b) transgenic lines. Total protein extracted from siliques was analysed using anti-FLAG antibody. Asterisks indicate non-specific bands. c, Comparison of mature dry seeds of wild-type (WT), tfl1-20, gDN, TFL1:DN and tfl1-20 TFL1:DN plants. Scale bar, 500 µm. d, Comparison of the peripheral endosperm development of cleared whole-mount seeds of WT, tfl1-20, gDN, TFL1:DN and tfl1-20 TFL1:DN at 4 DAP by confocal microscopy. The auto-fluorescence (green fluorescence) signal was generated by glutaraldehyde treatment. Arrowhead indicates new cell wall during endosperm cellularization. Merge, merge of green fluorescence and bright field images. Scale bars, 50 µm. The experiments in a–d were repeated three times independently with similar results. e, Percentage of seeds with syncytial or cellularized peripheral endosperm in WT, tfl1-20, gDN, TFL1:DN and tfl1-20 TFL1:DN at 4 DAP examined by confocal microscopy. Randomly selected seeds from more than 10 siliques for each genotype were examined.

Source data

Extended Data Fig. 9 tfl1-20 gTFL1-3HA TFL1:3FLAG-DN-ran2 and tfl1-20 gTFL1-GFP TFL1:3FLAG-DN-ran2 produce large seeds like tfl1-20.

a, Comparison of mature dry seeds of WT, TFL1:3FLAG-DN-ran2 (TFL1:DN), tfl1-20 gTFL1-3HA TFL1:DN and tfl1-20 gTFL1-GFP TFL1:DN plants. Scale bar, 500 µm. b, Quantitative analysis of seed size parameters of different genotypes. Box plots show medians (lines), interquartile ranges (boxes), and whiskers (extending 1.5 times the interquartile ranges) of seed size parameters (area, perimeter, length, and width) of seeds of different genotypes (WT, n = 129; TFL1:DN, n = 110; tfl1-20 gTFL1-3HA TFL1:DN, n = 118; tfl1-20 gTFL1-GFP TFL1:DN, n = 105). Percentage change (%) in a seed parameter of a specific genotype is shown relative to the mean value of WT plants set as 100%. The increase in the percentage in a specific genotype over WT is indicated above each box. Asterisks indicate significant differences between WT plants and other genotypes (two-tailed Mann-Whitney test, P < 0.0001). P values are all less than 1 × 10−15. c, Localization of TFL1-GFP in another tfl1-20 gTFL1-GFP TFL1:DN seed at 4 DAP by cryosectioning. TFL1-GFP signal is observable in the chalazal endosperm (CZE; left and middle panels) and its close-up view (right two panels). Note that TFL1-GFP is absent in all the syncytial peripheral endosperm (SPE). Merge, merge of GFP and bright field images. Scale bars, 40 μm (left) and 10 μm (right). The experiments in a,c were repeated three times independently with similar results.

Source data

Extended Data Fig. 10 Examination of seed size phenotypes and gene expression pertaining to ABI5.

a, Quantitative analysis of seed size parameters of tfl1-20, abi5-1, and tfl1-20 abi5-1. Box plots display medians (lines), interquartile ranges (boxes), and whiskers (extending 1.5 times the interquartile ranges) of seed size parameters (area, perimeter, length and width) of seeds of different genotypes (WT, n = 198; tfl1-20, n = 133; abi5, n = 110; tfl1-20 abi5, n = 138). Percentage change (%) in a seed parameter of a mutant is shown relative to the mean value of wild-type (WT) plants set as 100%. The increase in the percentage in a mutant over WT is indicated above each box. Asterisks indicate significant differences between WT plants and other mutants (two-tailed Mann-Whitney test, P < 0.001). P values for seed area, perimeter, length, and width between WT and abi5 are 1.48 × 10−12, 1.74 × 10−8, 1.55 × 10−11, and 1.78 × 10−4, respectively, while P values between WT and tfl1-20 or tfl1-20 abi5 are all less than 1 × 10−15. b, Quantitative analysis of ABI5 expression in various tissues (upper panel) or developing siliques at 2 to 7 days after pollination (DAP, lower panels). OF, open flower; IS, inflorescence stem; RL, rosette leaf; Rt, root; Sil, silique; CL, cauline leaf; FB, flower bud. Results were normalized against the expression levels of the U-BOX gene as an internal control. Expression levels in the lower panel are shown as relative values to the 2 DAP level set as 1. Values are mean ± SD of three biological replicates. c, Comparison of GUS staining signals between gABI5-GUS and tfl1-20 gABI5-GUS seeds at 4 DAP. The gABI5-GUS construct was generated through translational fusion of a GUS gene with a 4.1-kb ABI5 genomic fragment before the stop codon TAA. Scale bar, 50 µm. The experiment was repeated three times independently with similar results. d, Phylogenetic analysis of the A group of basic leucine zipper (bZIP) transcription factors. The phylogenetic tree was generated by MEGA4 using the Neighbor-joining algorithm. Numbers on the branches indicate bootstrap values in 1,000 replicates. ABI5 and FD are marked with red dots.

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Zhang, B., Li, C., Li, Y. et al. Mobile TERMINAL FLOWER1 determines seed size in Arabidopsis. Nat. Plants 6, 1146–1157 (2020). https://doi.org/10.1038/s41477-020-0749-5

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