jipb13193-sup-0001-Li_SupplementalData.pdf8.3 MB Figure S1. Expression patterns of stomatal lineage markers in wild-type and flp-1 xs01c mutant cotyledons. (A, B) SPCH:SPCH-GFP expression patterns are similar between Col and flp-1 xs01c (2 DAP). (C, D) Expression of GMC maker MUTE:MUTE-GFP can be found in the cells within the tumors of flp-1 xs01c (3 DAP). (E, F) Expression of E1728 indicates the mature GCs that occasionally formed within the tumors of flp-1 xs01c (8 DAP). Scale bars, 20 µm. Figure S2. The mutation in FBS4 splicing site leads to reading frame shifts. (A) The point mutation in the second intron of FBS4 results in two different splice variants. The first wrong cut (FBS4-WC-1), 588 bp-601 bp of FBS4 cDNA sequence is deleted. The second (FBS4-WC-2), the second intron is not spliced successfully. Two splice variants all lead to frame shift mutation. (B) The gene expression levels of FBS4 in fbs4-1 higher than Col, primers are shown by red arrows (A). Asterisks indicate significant differences relative to Col (Student's t-test, ** P<0.01). Data are means ± SD (n=15). Figure S3. No significant changes on stomatal density and index in fbs4-1 and fbs4-2 mutants (A) The stomatal densities. (B) The stomatal indexes. Data are represented as means ± SD (n=25). No significant differences in comparison with Col after Student's t-test. Figure S4. Transformation of FBS4 gene complements the stomatal defects in flp-1 fbs4-1 mutants. (A, B) In FBS4:GFP-gFBS4 transgenic line#25 and #60, transformation of genomic FBS4 partially complements the stomatal clustering phenotype in flp-1 fbs4-1. (C) In transgenic line#41, FBS4 fully complements the stomatal defects of flp-1 fbs4-1 to normal. Stomatal GCs are highlighted with blue color. Scale bars, 20 µm. (D) The transcriptional level of FBS4 in transgenic line#41 is much higher than other lines, corresponding to its stronger complementary effect on stomatal defects. The stomatal phenotype of Line#21 is shown in Figure 1N. (E) Quantitative analysis the cluster size. Data represent the mean ± SD (n=25). Figure S5. Additional mutation of FBS4 causes retarded plant growth in flp-1 mutant background (A) The side-views of 30-day-old plants. Addition mutation of FBS4, either by fbs4-1 or by fbs4-2, caused a retarded plant growth and delayed flowering in flp-1 mutants. Transformation of FBS4:GFP-gFBS4 restored the plant growth of flp-1 fbs4-1 (transgenic line#20). (B) Topviews of same seedlings shown in panel (A). Scale bar, 2 cm. Figure S6. The sequence alignments of FBS family genes and proteins. (A) Alignment of FBS1, FBS2, FBS3, FBS4 cDNA sequences. Black underline indicates the region encoding F-BOX domain. (B) Alignment of amino acid sequences of FBS family proteins. Black underline indicates the F-box domain, in which P85 of FBS1 and P166 of FBS4 (indicated by red asterisks) are conserved residues for their interaction with ASK of SCF complex. Figure S7. Generation and identification of FBS family mutants. (A) fbs1-2 CRISPR was created by CRISPR/Cas9-based genome editing. PAM sequence is labeled in green color. (B) Identification of T-DNA insertion lines fbs2-1 and fbs3-1. Primers used in RT-PCR are indicated by red arrows. Figure S8. Phylogenetic relationships of FBS proteins in plants. Phylogenetic tree was constructed using amino acid sequences by the neighbor-joining method of MEGA7. Bootstrap Values for 1,000 replicates are given in nodes as percent. Arabidopsis thaliana, Zea mays, Brachypodium distachyon, Glycine max, Oryza sativa, Physcomitrella patens, Zostera marina, Medicago truncatula, Sorghum bicolor, Solanum lycopersicum. Underlined letters used for the species abbreviations. Figure S9. fbs2 and fbs3 mutants are hypersensitive to salt stress. (A-F) Comparing to Col, both fbs2-1 and fbs3-1 mutants display hypersensitive response to salt stress. Top-view images of 20-day-old seedlings after 6 days treatment with 300 mM NaCl. (G, H) fbs4-1 displays a similar sensitivity to salt stress with Col. (I) Quantitative analysis of seedling survival rate. Data represent the mean ± SD (3 individual experiments, n=64); Statistical analysis, P<0.05, Duncan's multiple range test. Figure S10. FBS4 is involved salt stress and JA-responsive gene expression. (A-H) Top-view images of 20-day-old seedlings after 6 days of 300 mM NaCl treatment. Comparing to Col or fbs4-1, flp-1 displays a sensitive response to salt stress, while flp-1 fbs4- 1 seedlings are hypersensitive to salt stress. (I) Quantitative analysis the survival rates. Data represent the mean ± SD (3 individual experiments, n=64). The specific differences in means were compared with Duncan's multiple range test (P<0.05). (J) Real-time quantitative PCR analysis represent the relative expression changes of 3 JA responsive genes after 50 μM MeJA treatment in 8-DAP plants, JAZ10 (at 1 h), VSP2 (at 8 h), and PDF1.2 (at 24 h). The mRNA levels were normalized to ACTIN8. Data represent the mean ± SD (repeated from 3 biological experiments, n=12). Asterisks indicate significant difference to Col (Student's t-test, **P<0.01, *P<0.05). Figure S11. FBS4 is predicated on having a preferential expression profile within stomatal lineage cells. The results obtained from Arabidopsis eFP browser website (http://bar.utoronto.ca/). Figure S12. Expression pattern of FBS4:GUS reporter in Arabidopsis. (A) FBS4:GUS expression in a 5-DAP seedling. (B) GUS expression in veins and epidermis of cotyledon. (C) Preferential expression of FBS4:GUS in stomatal lineage cells in cotyledon epidermis. (D) GUS expression in a lateral root primordium. (E) GUS expression in a primary root tip. (F) GUS expression in vein and stomata in a hypocotyl. (G) No GUS signal is found in a 25-DAP rosette leaf. (H) GUS in 25-DAP inflorescence and young siliques. (I) No GUS expression in a mature capsule. Scale bars, 2 mm (A, G, H, I), 200 µm (B), 50 µm (D, E), and 20 µm (C, F). Figure S13. Subcellular localization of FBS4 in stomatal precursors. (A) Confocal images of epidermis of FBS4:GFP-gFBS4 cotyledons. Cell outlines are counterstained with propidium iodide but presented in purple color. Due to the weak expression of GFP-gFBS4, image brightness of GFP channel was upregulated before merging. xy view, a single optical section from confocal z-stacks. yz view and xz view, the orthogonal views along the yellow crossline. (B) The enlarged three dimensional images of the region within the white box shown in panel (A). GFP signals are enriched in the cytoplasm and the nucleus of a stomatal precursor. Scale bars, 20 µm (A), 5 µm (B). Figure S14. F-box domain mediates FBS4 interaction with ASK1. (A) Negative control for yeast two hybrid assay shown in Figure 4A. Transformants grown on the control media lacking His, Trp and Ura (SD/-His/-Trp/-Ura, -3) and selective media lacking His, Leu, Trp and Ura (SD/Gal/Raf/-His/-Leu/-Trp/-Ura, -4) with different dilution times (10x, 100x, 1000x). (B) The interaction between FBS4 and ASK1 was verified as well by a luciferase complementation imaging (LCI) assay. But, mutated FBS4ΔF-box failed to interact with ASK1, confirming that the F-box domain is essential for the FBS4-ASK1 interaction. Figure S15. The FBS4 transcriptional levels in transgenic plants. The relative FBS4 gene transcriptional levels in multiple transgenic lines were checked by RT-qPCR, comparing with the FBS4 transcript in non-transformed Col plants. Data represents the mean ± SD (n = 16). No significant difference of FBS4 expression is found between line#11, line#4, and line#5 after Student's t-test statistical analysis. Figure S16. Quantitative analysis of FBS4 expression level and phenotype in FBS4:gFBS4 flp-1 line#17 transgenic plants. (A) The FBS4 transcriptional levels in flp-1 and FBS4:gFBS4 flp-1 transgenic line#17 were checked by RT-qPCR. (B) Quantitative analysis of the cluster size in flp-1 and FBS4:gFBS4 flp-1 line#17. Comparing with line#4 (see Figure 4G), the effect of FBS4 expression on the formation of stomatal clusters in flp-1 is limited in line#17. Data represents the mean ± SD (n = 25). Figure S17. Generation of CYCA2;3 CRISPR mutant. Diagram showing the target and mutated sites of cyca2;3 CRISPR by CRISPR/Cas9 technology. The gRNA sequences within the second exon of CYCA2;3 was indicated with underline. PAM sequences were labeled in green. Figure S18. The proteasome inhibitor MG132 stabilizes CYCA2;3-GFP in root tips. (A) The GFP signals in 5 DAP root tip of XVE:CYCA2;3-GFP after induction. (B) Application of 26S Proteasome Inhibitor MG132 increases the GFP signals in a root tip of XVE:CYCA2;3-GFP. Green, GFP; Purple, cell outlines are counterstained with propidium iodide. Scale bars, 50 μm. Figure S19. Quantitative and statistical analysis of the fluorescent intensities of CYCA2;3-GFP that coexperssed with FBS4. Coexpression with MYC-FBS4, but not with mutated MYC-FBS4 △F-box , dramatically reduced the fluorescence intensity of CYCA2;3-GFP, relative to the intensity level of CYCA2;3-GFP coexpressed with MYC. The experiment was repeated three times with similar results. Data are means ± SD (n = 30). Asterisks indicate the significant difference after Student's t-test, **P<0.01. Figure S20. Quantitative analysis of CYCA2;3-GFP fluorescent intensity in roots. (A) Relative intensity of CYCA2;3-GFP signals in the epidermal cells of elongation zone in Col and fbs4-1 roots. (B) Relative intensity of CYCA2;3-GFP signals in the root caps of Col and fbs4-1. The experiment was repeated three times with similar results. Data are means ± SD (n=46). Asterisks indicate significant differences between Col and fbs4-1 (Student's t-test, **P<0.01). Figure S21. DNA ploidy distribution profiles in Col and fbs4-1 cotyledons. (A, B) Flow cytometric analysis of 11 DAP Col and fbs4-1 cotyledons. (C) Quantitative analysis DNA ploidy levels. Figure S22. FBS4 is not involved in root length regulation. Length of 6-DAP root meristematic zone. Additional mutations of fbs4-1 and flp-1 has no effect on the root meristematic zone length in uvi4. Data are means ± SD (n ≥ 15). Letters above the columns indicate the statistic analysis results after Duncan's multiple range test (P<0.05). Table S1. List of primers used in this study.