Volume 23, Issue 6, Pages 1879-1890 (May 2018) FTIP-Dependent STM Trafficking Regulates Shoot Meristem Development in Arabidopsis  Lu Liu, Chunying Li, Shiyong Song, Zhi Wei Norman Teo, Lisha Shen, Yanwen Wang, David Jackson, Hao Yu  Cell Reports  Volume 23, Issue 6, Pages 1879-1890 (May 2018) DOI: 10.1016/j.celrep.2018.04.033 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 FTIP3 and FTIP4 Are Essential for Shoot Meristem Development (A and B) Comparison of 35-day-old wild-type (A) and ftip3-1 ftip4-1 (B) (upper panel, side view; lower panel, top view) plants. The ftip3-1 ftip4-1 mutant displays the dwarf and bushy phenotype. The arrow indicates the main inflorescence apex. Scale bars, 1 cm. (C and D) Comparison of main inflorescence apices of 35-day-old wild-type (C) and ftip3-1 ftip4-1 (D) plants. Scale bars, 2 mm. (E and F) Scanning electron micrograph analysis of main inflorescence shoot apices of wild-type (E) and ftip3-1 ftip4-1 (F) plants. The inflorescence apical meristem is highlighted in red, and young flowers in stage 1 to 5 are indicated. Scale bars, 100 μm. (G and H) Median longitudinal sections of main inflorescence shoot apices of wild-type (G) and ftip3-1 ftip4-1 (H) plants. Scale bars, 100 μm. (I and J) Transverse sections of main inflorescence stems of wild-type (I) and ftip3-1 ftip4-1 (J) plants. Scale bars, 100 μm. See also Figures S1 and S2. Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Localization of FTIP3 and FTIP4 (A and B) Confocal analysis of FTIP3-RFP (A) and FTIP4-GFP (B) protein distribution in SAM cryosections from 35-day-old gFTIP3-RFP ftip3-1 ftip4-1 and gFTIP4-GFP ftip3-1 ftip4-1 lines, respectively. Scale bars, 25 μm. (C) Confocal analysis of subcellular localization of FTIP4-GFP in SAM cells of 35-day-old gFTIP4-GFP ftip3-1 ftip4-1. GFP, GFP fluorescence; DAPI, DAPI fluorescence; BF, bright field; merge, merge of GFP, DAPI. and bright-field images. Scale bars, 10 μm. (D–G) Analysis of 4HA-FTIP4 localization in SAM cells of 35-day-old 4HA-gFTIP4 ftip3-1 ftip4-1 by immunogold electron microscopy. 4HA-FTIP4 localizes in intracellular vesicles (IVs) (D and E) and the plasma membrane (F), but not in the nucleus (N) (G). CW, cell wall. Arrowheads indicate the location of gold particles. Scale bars, 500 nm (D–F) and 1 μm (G). See also Figure S3. Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 FTIP3 and FTIP4 Interact with STM (A) Yeast two-hybrid assay of the interaction between STM and the N-terminal region of FTIP3 (aa 1–480; N480) or FTIP4 (aa 1–481; N481). Transformed yeast cells were grown on SD-Ade/-His/-Leu/-Trp medium. (B) BiFC analysis of the interaction between STM and FTIP4. EYFP, enhanced yellow fluorescent protein; H2B-RFP, the fluorescent nuclear reporter (core histone 2B fused to red fluorescent protein); BF, bright field; merge, merge of EYFP, H2B-RFP, and bright-field images. Scale bars, 20 μm. (C) In planta interaction between FTIP4 and STM shown by coimmunoprecipitation. Protein extracts from tobacco leaves expressing 35S:STM-9myc alone or both 35S:STM-9myc and 35S:4HA-FTIP4 were immunoprecipitated by anti-HA agarose. The input and coimmunoprecipitated proteins were detected by anti-myc and anti-HA antibody. (D) In vivo BiFC analysis of the interaction between STM and FTIP4 driven by their own promoters in cells in the peripheral region of SAMs of 35-day-old transgenic Arabidopsis plants. EYFP, enhanced yellow fluorescent protein; merge, merge of EYFP and DAPI images. Scale bars, 5 μm. (E–J) The meristem defect in stm-10 is enhanced by ftip3-1 ftip4-1. At 9 days after germination, wild-type (E), ftip3-1 ftip4-1 (F), and a weak stm-10 mutant (G) generate two rosette leaves. In contrast, the meristem of stm-10 ftip3-1 ftip4-1 (H) terminates without generating leaf, which is similar to the phenotype exhibited by a null stm-11 mutant (I) or stm-11 ftip3-1 ftip4-1 (J). Scale bars, 0.5 cm. (K) Quantification of the phenotypic enhancement in stm-10 ftip3-1 ftip4-1. See also Figure S4. Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 FTIP3 and FTIP4 Mediate STM Trafficking in the SAM (A and B) Confocal analysis of YFP-STM protein distribution in SAM cryosections from 35-day-old STM:YFP-STM plants in wild-type (A) and ftip3-1 ftip4-1 (B) backgrounds. Scale bars, 25 μm. (C and D) Comparison of YFP-STM signal intensity in the apical region of wild-type (C) and ftip3-1 ftip4-1 (D) plants shown in (A) and (B). The color code (blue to red) depicts YFP-STM signal intensity (low to high). Rectangular boxes mark the regions for signal quantification shown in (E). Scale bars, 25 μm. (E) Measurement of YFP-STM intensity profiles using ImageJ along the horizontal axis of the rectangular boxes depicted in (C) and (D). The similar results were observed in median longitudinal cryosections of 10 independent plants for each genotype. (F) Confocal analysis of YFP-STM localization in cells in the peripheral region of SAMs of 35-day-old STM:YFP-STM plants in wild-type and ftip3-1 ftip4-1 backgrounds. DAPI staining labels nuclei, while white lines mark the cells for signal quantification shown in (G). YFP, YFP fluorescence; DAPI, DAPI fluorescence; BF, bright field; Merge, merge of GFP, DAPI, and BF images. Scale bars, 5 μm. (G) Measurement of fluorescence intensity profiles of YFP-STM and DAPI using ImageJ along the lines on the selected wild-type and ftip3-1 ftip4-1 SAM cells in (F). Peak areas with DAPI intensity of more than 50 indicate the nucleus regions. (H and I) Quantification of relative fluorescence intensity (H) or relative intensity in nucleus versus cytoplasm (I) of YFP-STM signals in cells in the peripheral region of SAMs from confocal images generated from 10 independent plants each for STM:YFP-STM or ftip3-1 ftip4-1 STM:YFP-STM as shown in (F). Fluorescence intensity is indicated as the mean gray value representing the average signal intensity in selected regions, including nucleus, cytoplasm, and both nucleus and cytoplasm (total). Data represent mean values ± SD (n = 70; ∗∗p < 0.001; ∗∗∗p < 0.05). See also Figure S5. Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 FTIP4 Affects STM Subcellular Localization in Tobacco (N. benthamiana) Leaf Epidermal Cells (A) GFP-STM localizes in the plasma membrane, cytoplasmic vesicles, and nucleus labeled by H2B-RFP. Scale bar, 10 μm. (B) Coexpression of 35S:GFP-STM and 35S:FTIP4 results in a marked decrease in GFP-STM signals in the plasma membrane but an increase in GFP-STM signals in cytoplasmic vesicles. Scale bar, 10 μm. (C) Coexpression of 35S:GFP-STM and 35S:RFP-ARA6 shows substantial colocalization of GFP-STM and the endosome marker RFP-ARA6 in the plasma membrane and endosomes. Scale bar, 10 μm. (D) Coexpression of 35S:GFP-STM, 35S:FTIP4 and 35S:RFP-ARA6 results in a marked decrease in GFP-STM signals in the plasma membrane but an increased colocalization of GFP-STM and RFP-ARA6 in the endosomes. Scale bar, 10 μm. (E) Coexpression of 35S:FTIP4-GFP and 35S:RFP-STM shows colocalization of FTIP4-GFP and RFP-STM in endosomes. Arrowheads and arrows indicate nuclei and endosomes, respectively. GFP, GFP fluorescence; RFP, RFP fluorescence; BF, bright field; merge, merge of GFP, RFP, and bright-field images. Scale bar, 10 μm. See also Figure S6. Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 FTIP3 and FTIP4 Regulate STM Trafficking (A) Analysis of 3FLAG-STM distribution in the cell wall of cells located at the periphery of SAMs of 35-day-old STM:3FLAG-STM plants in wild-type and ftip3-1 ftip4-1 backgrounds by immunogold electron microscopy using anti-FLAG antibody. CW, cell wall; IS, intercellular space; PD, plasmodesma. Arrowheads indicate gold particles near or in plasmodesmata in the cell wall. Scale bars, 500 nm. (B) Control experiments showing immunogold electron microscopy analysis of gold particle distribution in the cell wall of cells located at the periphery of SAMs of 35-day-old STM:3FLAG-STM (using mouse immunoglobulin G [IgG] antibody; upper panel) or wild-type (using anti-FLAG antibody; lower panel) plants. Scale bars, 500 nm. (C) Frequency histograms of the appearance of 3FLAG-STM immunogold signals or background signals in shoot meristem cells from sections (as shown in A and B) probed with anti-FLAG or mouse IgG antibody (n = 70). (D) Quantification of 3FLAG-STM immunogold signals or background signals in shoot meristem cells from sections (as shown in A and B) probed with anti-FLAG or mouse IgG antibody (n = 70 ± SD). Asterisk indicates a statistically significant difference (two-tailed paired Student’s t test, p < 0.001). (E) Confocal analysis of GFP-STM-2NLS protein distribution in the SAMs of 35-day-old STM:GFP-STM-2NLS plants in wild-type and ftip3-1 ftip4-1 backgrounds. Rectangular boxes mark the regions for quantification of signal intensities shown in (F). Scale bars, 25 μm. (F) Quantification of GFP-STM-2NLS signal intensities along the horizontal axis of rectangular regions depicted in (E) using the ImageJ plot profile. The similar results were observed in the SAMs of 10 independent plants for each genotype. (G) Phenotypic comparison of 35-day-old ftip3-1 ftip4-1 and STM:GFP-STM-2NLS ftip3-1 ftip4-1 plants. Scale bar, 1 cm. See also Figure S7. Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 FTIP3 and FTIP4 Coordinate STM Trafficking to Mediate Maintenance and Differentiation of Shoot Stem Cells (A) In the cells in the peripheral region of wild-type SAMs, FTIP3 and FTIP4 recruit STM in endosomes and prevent STM trafficking to the plasma membrane. This allows BLH proteins to guide more STM protein to the nucleus, thus maintaining an appropriate frequency of differentiation of inflorescence shoot stem cells, which results in a wild-type inflorescence structure. (B) In the absence of FTIP3 and FTIP4, STM traffics frequently to the plasma membrane. This facilitates more intercellular trafficking of STM but decreases its localization in the nucleus, resulting in compromised stem cell maintenance and early termination of the main inflorescence shoot apex, as well as consequential dwarf and bushy phenotypes of ftip3-1 ftip4-1. Green arrows in right panels indicate proliferating inflorescence shoot apices. ER, endoplasmic reticulum; MVB, multivesicular body; PD, plasmodesma; TGN, trans-Golgi network. Cell Reports 2018 23, 1879-1890DOI: (10.1016/j.celrep.2018.04.033) Copyright © 2018 The Author(s) Terms and Conditions