Supplemental Data. Di Giorgio et al. (2016). Plant Cell /tpc

Transcription

1 Supplemental Figure 1. Synteny analysis of NIP4;1 and NIP4;2. Examination of the NIP4;1-NIP4;2 region in rabidopsis thaliana and equivalent evolutionary regions in other dicot genomes are shown on the left panel. Phylogenetic relationships among plants and. thaliana NIP4;1-NIP4;2 deduced from syntenic analysis are indicated in the right panel. Genomes (putative QP loci): rabidopsis thaliana (T5G37810, T5G37820), rabidopsis lyrata (Loc330314, Loc891407), apsella rubella (arubv m, arubv m), rassica rapa (rapara025437, rapara025436, rapara02543), Thellungiella halophila (Thhalv m, Thhalv m, Thhalv m), arica papaya (evm.model.supercontig_65.137), Theobroma cacao (Thecc1EG019213t1), Vitis vinifera (GSVIVT ) and Solanum tuberosum (PGS0003DMP ). PF00230: QP protein, PF00010: helix-loop-helix DN-binding domain containing protein, PF00069: protein kinase family protein, PF00036: Osam2 - almodulin, PF08543: kinase, pfk family, PF00403: heavy-metal-associated domain-containing protein, PF06886: Targeting protein for Xklp2, PF00035: Double-stranded RN binding motif, F00226: dnaj domain containing protein, PF oxoprolinase. 1

2 T4G19030 (NIP1;1) T4G18910 (NIP1;2) T2G34390 (NIP2;1) T1G31885 (NIP3;1) T5G37810, T5G37820 (NIP4;1, NIP4;2) T4G10380 (NIP5;1) T1G80760 (NIP6;1) T3G06100 (NIP7;1) T3G61430 (PIP1;1) T2G45960 (PIP1;2) T1G01620 (PIP1;3) T4G00430 (PIP1;4) T4G23400 (PIP1;5) T3G53420 (PIP2;1) T2G37170, T2G37180 (PIP2;2, PIP2;3) T5G60660 (PIP2;4) T3G54820 (PIP2;5) T2G39010 (PIP2;6) T4G35100, T2G16850 (PIP2;7, PIP2;8) T3G04090 (SIP1;1) T5G18290 (SIP1;2) T3G56950 (SIP2;1) T2G36830 (TIP1;1) T3G26520 (TIP1;2) T4G01470 (TIP1;3) T3G16240 (TIP2;1) T4G17340 (TIP2;2) T5G47450 (TIP2;3) T1G73190 (TIP3;1) T1G17810 (TIP3;2) T2G25810 (TIP4;1) T3G47440 (TIP5;1) # of samples Supplemental Figure 2. Expression patterns of rabidopsis thaliana aquaporin genes. Heat map of normalized absolute expression values of 35 rabidopsis thaliana QP genes obtained using GENEVESTIGTOR ( Pollen-specific QPs are in red and QPs expressed in pollen and also in sporophytic tissues are in blue (107 microarrays consulted). 2

3 D x NIP4;1 pro :GUS NIP4;1 pro :GUS x x NIP4;2 pro :GUS NIP4;2 pro :GUS x E F G H I NIP4;1 pro :GUS J K L M N NIP4;2 pro :GUS O P Q R S 5 days 10 days Supplemental Figure 3. GUS staining of pistils and seedlings. GUS staining of pistils crossed with transgenic NIP4;1 pro :GUS () and NIP4;2 pro :GUS () pollen, and reciprocal crosses (,D). Transgenic NIP4;1 pro :GUS (E-I) and NIP4;2 pro :GUS (J-N) seedlings grown for 5 and 10 days in 0.8% agar supplemented with asta 12.5 mg/l. plants were used as control (O-S). 3

4 D E F Supplemental Figure 4. Pollen bombardment assays. onstructs EGFP-NIP4;1 (,), EGFP-NIP4;2 (,D) and LT52 pro :RFP-LT52 pro :ami356 (E,F) were validated by tobacco pollen bombardment and epifluorescence microscopy. White arrows indicate positive pollen tubes (,D) and a pollen grain (F). ar = 200 µm. 4

5 F 60min 60min 1min G 10min D 60 min H 20min E 60min 60min I J K Supplemental Figure 5. Disruption of EGFP-NIP4;1 cycling. (-H) rabidopsis pollen tubes were germinated for 30 min in PGM and then treated with 3 µg/ml F before imaging using a confocal microscope for up to 60 min. Representative fluorescence confocal images (left panels) and differential interference contrast (DI) optics (right panels). ar = 10 µm. () Effect of F treatment for 60 min in a pollen tube, showing the formation of a bubble tip as control of growth arrest. (-E) Effect of F treatment on EGFP- NIP4;1 pollen tubes (line /NIP4;1-20) after 1 (), 10 (), 20 (D) and 60 min (E). (F) Effect of F treatment for 60 min in a grain used as control. (G) Effect of F treatment for 60 min on EGFP-NIP4;1 mature pollen. (H) Effect of F treatment for 60 min on EGFP-NIP4;2 mature pollen, used as negative control. (I-K) rabidopsis pollen tubes were germinated for 30 min in PGM and then treated with 1 µm FM4-64 and either with 6 µg/ml F or 100 µm 23. Fluorescence confocal images of GFP and FM4-64 signals and DI are shown. ar = 10 µm. (I) GFP and FM4-64 signal in a LT52 pro :EGFP-NIP4;1 pollen tube as control. (J) Effect of F treatment on a EGFP-NIP4;1 pollen tube after 25 min. (K) Effect of 23 treatment on a EGFP-NIP4;1 pollen tube after 10 min. total of 13 independent germinations analyzing at least 20 grains and pollen tubes for each genotype and condition were performed. 5

6 La1 La1 1 3 RP038 RP007 LP038 LP007 LP013 2 RP013 La1 tnip4;1 L1 RP835 2 LP835 LP142 1 RP142 La1 tnip4;2 DN ladder (kb) nip4;1-1 nip4;1-2 nip4;1-3 nip4;2-1 nip4; Supplemental Figure 6. Single T-DN nip4;1 and nip4;2 mutant lines. Schematic diagram of NIP4;1 () and NIP4;2 () genes. lack boxes represent exons, and predicted promoter and 5 and 3 UTRs are shown as grey boxes. T-DN insertions are indicated with different colored triangles. Right (RP) and Left (LP) primers used for genotyping are indicated with arrows using the same colors as the triangles. Left border primer (L) is also shown. () Genotyping PRs were performed from genomic DN (gdn) from homozygous mutant plants nip4;1-1, nip4;1-2, and nip4;1-3 and nip4;2-1 and nip4;2-2, using line-specific sets of RP, LP and L primers. gdn was used as control in each PR. 6

7 cdn E1 E2 E3 E4 E5 ami260 ami356 Target genes NIP4;1 & NIP4;2 5 3 / : amirn 260: Target genes NIP4;1 & NIP4;2 5 3 / : amirn 356: TGGTTGTTTTTTG GTTGGTGT **** ************** GGTTTGTTTGGTG TGGTTTTT ******************* L Pf amirn ami- KanR T35S RFP LT52pro LT52pro S T35S ami-s Pr 260/356 II R Pf amirn-pr 260 II gdn cdn water Pf amirn-pr 356 II gdn cdn water 313 bp Supplemental Figure 7. Double knockdown amirn lines. () Schematic description of the hypothesized duplexes formed by interactions between NIP4,1 and NIP4;2 genes and the two amirns (ami260 and ami356). Paired bases are indicated by asterisks. () Schematic diagram of binary plasmid pk7wg2d expressing amirn sense (ami-s) and antisense (ami-) sequences. The LT52 pro :RFP reporter gene was included in the construct to identify pollen carrying the amirns. Forward (Pf amirn) and reverse (Pr 260 II and 356 II) primers used for genotyping PR and RT-PR assays are indicated with arrows. () Genotyping PRs with genomic DN (gdn) and RT-PR assays (cdn) from double knockdown ami260-4 and ami356-1 plants, using line-specific primers. gdn and cdn were used as control. 7

8 D E F G H I J Supplemental Figure 8. Double knockdown amirn lines expressing the RFP reporter gene. (, D, G, H) right-field images. (, E, I, J) Epifluorescence images showing nuclei stained with Hoechst (blue filter), and insets in E and J showing the three nuclei in detail. (, F) Epifluorescence images showing RFP expression (red filter). (-) Mature pollen grain of the ami260-4 line. ar = 20 µm. (D-F) Pollen tube of the ami356-1 line germinated in vitro for 4h. ar = 50 µm. pollen grain (G, I) and tube (H, J) were included as controls. ar = 20 µm (G,I) and 50 µm (H-J). 8

9 D E F Supplemental Figure 9. SEM images of pollinated pistils and developing seeds. (-D) Pollinated pistils from () and double knockdown qrt (), ami356-1 () and ami260-4 (D) opened flowers (stages late 13 and 14). Yellow arrows indicate pollen grains with defects in pollen rehydration showing a collapsed shape. (E-F) Developing seeds in manually opened siliques of (E) and ami356-1 (F) plants. Yellow arrow indicates an unfertilized ovule. 9

10 ami260-4 nip4;1-2 D nip4;2-2 Supplemental Figure 10. Pollen microgametogenesis. Development of (), double knockdown ami260-4 (), single mutant nip4;1-2 () and nip4;2-2 (D) pollen grains. Nuclei were stained with Hoechst Insets show a representative pollen grain in detail. White arrows indicate bicellular pollen grains. ar = 200 µm. 10

11 D E F Supplemental Figure 11. Viability of mature pollen grains. Mature pollen was stained with FD for 10 min. Epifluorescence images of viable (green) and non-viable (blue) pollen grains of (), double knockdown ami260-4 () and ami356-1 () and single T-DN mutants nip4;1-3 (D), nip4;1-1 (E) and nip4;2-1 (F). White arrows show non-viable, collapsed pollen grains. ar = 50 μm. 11

12 ami260-4 ami356-1 nip4;1-3 nip4;2-1 Supplemental Figure 12. Pollen tube growth in self-crossed pistils. Decolorized aniline blue staining images of self-crossed pistils of (n=10), double knockdown (ami260-4, n=14 and ami356-1, n=17) and single mutant (nip4;1-3, n=8 and nip4;2-1, n=9) flowers at stage 15 post-fertilization. Only pistils with completely pollinated stigmas were analyzed. White asterisks indicate non-fertilized ovules. ar = 200 μm. 12

13 Pollen tube length (µm) ** * ns ns ns ns ami260-4 ami X 5X 10X Kl Pollen tube length (µm) ** * ns ns ami260-4 ami Temperature Pollen tube length (µm) ** * * ns ami260-4 ami M 0.08 M Glycerol Supplemental Figure 13. Effect of temperature, salt, and osmotic stress on pollen tube growth. (-) In vitro pollen germination of wild type () and amirn (ami260-4 and ami356-1) plants on standard or modified solid PGM. Mean tube length ± SEM of ami260-4/ and ami356-1/ pollen germinated in solid PGM with () standard (1X = 5 mm Kl) or increasing salt concentrations (5X and 10X), at () standard (22 ) or higher (28 ) temperature, and () without (0 M) or with 0.08 M glycerol. ssays were performed in duplicate and repeated twice; 100 pollen tubes were measured for each replicate. Data was analyzed by one-way NOV and Tukey s test, (*p<0.05, **p<0.01, ns: not significant). 13

14 D Supplemental Figure 14. Subcellular localization of EGFP-NIP4;1 and EGFP-NIP4;2 in Xenopus oocytes. Radial (x z) confocal images of Xenopus laevis oocytes expressing EGFP-NIP4;1 () EGFP-NIP4;2 (), EGFP- NIP4;1-S267 () and EGFP-NIP4;2-S267 (D) (green), previously injected with TMR-Dextran (red). ar = 10 µm. These are representative images of a total of three independent assays analyzing at least 20 eggs. 14

15 i) ii) Supplemental Figure 15. Functional assays of NIP4;1 and mutant NIP4;1-S267 in yeast () Water transport assays were done in yeast spheroplasts transformed with NIP4;1, NIP4;1-S267 or human QP8 as a positive control, prepared and mixed in a stopped flow apparatus with hyposmotic solution to induce cell swelling. Spheroplasts from yeast cells (Y4741) transformed with the pyedp60u empty vector or containing the indicated QP homologs, were suspended in 0.5 M sorbitol plus 0.4 M potassium sulfate at an OD 600 of 2.0 and mixed in a fast kinetics instrument with an equal volume of hypotonic buffer 0.5 M sorbitol at 10 (300 mosmol difference). Each line represents the average of measurements. Data points were collected each 500 µs for 3 s. Measurements from the first 2 s are displayed. Representative result of two independent experiments. () Yeast growth and survival on synthetic medium with different concentrations of H 2 O 2. Yeast strains YNVW1 ( dur3) (i) and mep1-3 (ii) transformed with the empty vector pyedp60u or pyedp60u containing the indicated QP homologs, were spotted at an OD 600 of 0.02 and 2 on medium containing various concentrations (0, 0.2, 0.4, 0.8, 1.2, 1.8, 2.4 mm) of H 2 O 2. Growth was recorded after 8 days at 28. ll data were duplicated in three independent experiments. () Yeast growth and survival on synthetic medium with different concentrations of urea. YNVW1 ( dur3) yeast mutants transformed with the pyedp60u empty vector or containing the indicated QP homologs, were spotted at an OD 600 of 2, 0.02 or on medium containing various concentrations of urea or arginine as sole nitrogen source and growth was recorded after 9 days at 28. ll data were duplicated in three independent experiments. 15

16 SUPPLEMENTL TLES Supplemental Table 1. Microgametogenesis of single mutant nip4;1 and nip4;2 and double knockdown pollen. Microgametogenesis TP P+UP Mean SEM Mean SEM a a 0.18 ami b b 0.98 ami b b 0.79 nip4; b b 0.63 nip4; ab ab 0.30 Mean percentages of tricellular (TP), and bicellular and unicellular (P+UP) ± SEM pollen grains of, double knockdowns ami356-1 and ami260-4 and single mutants nip4;1-2 and nip4;2-2. ssays were performed in 4 replicates (i.e., plants) for each genotype. For each replicate, 10 flowers and >100 pollen grains were analyzed. olumns with different letters indicate significant difference (p<0.05, one-way NOV, Tukey s test). ollapsed-shape grains were excluded from this analysis. 16

17 Supplemental Table 2. Viability and hydration capacity of mature pollen. FD Relative % lethality SEM staining volume SEM 2.32 a a 0.02 ami b b 0.04 ami b b 0.03 nip4; b b 0.01 nip4; b b 0.02 nip4; ab a 0.03 nip4; ab a 0.02 Mean lethality percentage and relative volume ± SEM of, double knockdowns ami356-1 and ami260-4, and single mutants nip4;1-1 and nip4;1-3 and nip4;2-1 and nip4;2-1. ssays were performed in 6 replicates (i.e. plants) per genotype. For each replicate 5-8 flowers incubated for 10 min with FD and 100 and 30 pollen grains were analyzed for viability and volume assays, respectively. Different letters indicate significant difference (p<0.05, one-way NOV, Tukey s test). 17