Supplementary Fig. 1

Transcription

1 SalI plt52-gfp-tr4-nos mp R Nco I XbaI amhi SmaI amhi TG GG TT G GG T G GGG T GG GT T TG EcoRV SacI HindIII SphI PstI SalI 6pb 725pb 255 pb LT 52 GFP R4 NOS Nco I XbaI amhi SmaI amhi TG GG TT G GG T G GGG T GG GT T TG HindIII SphI PstI SalI plt52-gfp-ttu1-nos 6pb LT 52 GFP mp R EcoRV 725pb 1384pb TU1 SacI 255 pb NOS SP6 6pb Lat 52 plt52-gfp-ttu6-p35s 725pb smrs-gfp 135pb TU6 mp R 255 bp p35s T7 Lat52GFP E Lat52GFPTU1 F Lat52GFPTU6 Supplementary Fig. 1

2 o Noc 45min o Noc 45min E F Ory.1 M 45min Ory.1 M 45min Supplementary Fig. 2

3 Pollen tube lenght (um) Pollen tube lenght (um) E F H Effect of 5uM Noc on the pollen tube grow th (Exp1) * o MSO Noc I Effect of 5uM Noc on the pollen tube grow th (Exp 2) o MSO Noc 15min 45min 15min 45min Time points Time points Supplementary Fig. 3

4 Frequency Frequency Pollen tube diameter (um) Effect of 5uM Noc on the pollen tube diameter Kurtosis MSO 45min 5.48 Noc 45min MSO 45min Noc 45min MSO 45min Noc 45min Pollen tube diameter ( m) Pollen tube diameter ( m) Supplementary Fig. 4

5 Normalized intensity (a.u.) MSO t MSO 1F P MSO 62F P Shank 1.2 NO 1. MSO t (s) Supplementary Fig. 5

6 Polle tube lenght (um) Pollen tube lenght (um) Pollen tube lenght (um) Pollen tube lenght (um) Ory.5 M 15min Ory.1 M 15min Effect of.5um Oryzalin on tube growth (Exp.1) ** 15 min 45 min 4 2 o 45min Oryz,5uM 45min o 15min Oryz,5uM 15min Effect of.5um oryzalin on pollen tube growth (Exp.2) o 45min * Oryz,5mM 45min 15 min 45 min o 15min Oryz,5mM 15min Effect of.1um Oryzalin on tube growth (Exp. 1) Effect of.1um Oryzalin on pollen tube growth (Exp. 2) E o 45min o 15min Oryz,1uM 45min Oryz,1uM 15min 15 min 45 min F o 45min o 15min Oryz,1mM 45min Oryz,1mM 15min 15 min 45 min Supplementary Figure 6

7 istance of vacuoles from tip PM (um) F F F o t Ory.1 M 3min 2min MSO 3min F Noc 3min F E Effect of.1um oryzalin-mso and 5uM Noc on vacuole distribution F * ** Ory 3min Noc 3min o Ory 15min MSO 3min * Supplementary Figure 7

8 SUPPLEMENTRY FIGURE LEGENS Supplementary Figure 1. MTs were not visualized in living pollen tubes. () plt52- GFPR4-NOS, () plt52-gfptu1-nos () plt52-gfptu6-p35s. Medial plane images of pollen tubes transiently transformed with plat52gfp shows that GFP is diffusely expressed in the cytoplasm (), plat52-gfptu1 (E) and plat52-gfptu6 (F) are also expressed in the cytoplasm but are not incorporated into MTs. Scale bar, 1 m. Supplementary Figure 2. Effects of 5 M Noc and.1 M Oryzalin (ory) on. (-) In control tubes are organized as long, longitudinally oriented bundles in the older parts of the tube, wheras in the tip only a diffuse fluorescence was observed (). Often, thick bundle of short, similar to the actin fringe (arrows) was observed (). (-) istribution of was not affected by 5 M Noc in the tip and in the shank. fter 45min incubation (arrows in indicate the actin fringe). (E-F) The overall distribution of F seemed also not to be affected in the presence of.1 M Ory after 45min incubation. Scale bar, 1 m. Supplementary Figure 3. Effect of MSO and Noc on pollen tube lenght. (-) ontrol, MSO and Noc-treated pollen tubes after 15 min. (-F) ontrol, MSO and Noc-treated pollen tubes after 45 min. (H-I) Tube lengths with and without Noc were compared. Scale bar, 1 m. Supplementary Figure 4. Effect of Noc on pollen tube diameter. () Polle tube lenghts in the presence of MSO and Noc were compared. (-) istribution frequency of diameter values in the presence of MSO or Noc. Supplementary Figure 5. FRP experiments in the shank PM in the presence of MSO or Noc. () PM in the shank was bleached (orange and blue ROIs. MSO t). (-) recovery was monitored (orange and blue ROI) until recovery was complete (62F P). () No difference in the amount of fluorescence recovered and in the rate of recovery was observed in MSO compared with Noc-treated pollen tubes. Scale bar, 1 m. Supplementary Figure 6. Oryzalin completely depolymerises MTs and affected pollen tube growth. (-).5 M and.1 M Ory completely depolymerizes Mts after 15min incubation. (-).5 M Ory inhibited pollen tube growth after 45min incubation in two experiments. (E-F).1 M Ory does not inhibit pollen tube growth in two experiments. Scale bar, 1 m. Supplementary Figure 7. Effect of Oryzalin, MSO and Noc on vacuole distribution pattern. (-) Ory.1 M affected vacuole distribution, leading lateral association of compartments () respect to o (). Moreover, vacuoles were isplaced toward to apical PM in the presence of Ory after 15min and 3min incubation (E). (-E) MSO and Noc did not alter vacuole distribution in the cytoplasm. Scale ar, 1 m.

9 SUPPLEMENTRY METHOS onstruction of GFP fusion proteins To generate GFP-TU1 construct,. thaliana TU1 cn (ecotype olumbia, rabidopsis biological Resource enter) was amplified by PR with Phusion High-Fidelity N polymerase (iolabs) using sense oligo 5 -GTGTGGGGGTTT-3 and antisense 5 - GGTTTTTTGTT-3. Restriction sites (underlined) were engineered in the oligonucleotides to facilitate subcloning as a PvuII-SacI fragment. fter digestion with PvuII and SacI, the purified fragment was ligated to puln-lt52-mgfp4 (by courtesy of Professor Erik Nielsen, epartment of Molecular, ellular and evelopment iology, University of Michigan, US) and digested with EcoRV-SacI. Ligation was done according to the N ligation kit instruction manual (Takara). To verify the correct orientation of the insert, control PRs were performed using sense primer designed on GFP sequence (5 -GTTTGT-3 ) and antisense primer, hybridizing with the NOS terminator (5 -GGGGGG-3 ). Sequencing analysis (MR Genomics, University of Padua) confirmed the insertion of TU1 and its amplification without errors (data not shown). To generate the LT52-GFPTU6 construct, the pollen-specific promoter Lat52 (Twell et al., 199) was amplified by PR with Taq polymerase, (sense oligo 5 - TTTGGTGTTGGGTTGG-3 ; antisense oligo 5 - GGGTGGTTTTTTTTTTTGGTGTGTG-3 ) from plt52-gfps65 (kindly provided by r T. Resch and Prof.. Touraev, Max F. Perutz Laboratories, epartment of Plant Molecular iology, Vienna University). Restriction sites (underlined) were engineered in the oligonucleotides to facilitate subcloning as a XhoI-amHI fragment. The purified fragment was ligated with psp72- GFPTU6 (kindly provided by Prof. T. Hashimoto, Graduate School of iological Sciences Nara Institute of Science and Technology, Ikoma, Japan) (Ueda et al., 1999) and digested with XhoI- amhi. Terminator p35s was excised as an XbaI-XbaI fragment from plt52-gfps65 and ligated with psp72-lt52-gfptu6 vector cut with XbaI. To verify the correct orientation of the insert, control PRs were performed using sense primer designed on TU6 (5 - GTGGTGTTTGG) and T7 antisense primer. Sequencing analysis (MR Genomics) confirmed correct insertion of the p35s terminator. ll plasmids were purified using QIGEN plasmid purification kit according to the QIGEN protocol.

10 ctin filament labelling The were detected in pollen tubes grown in K medium and then incubated in fixing solution, following the protocol described in Lovy-Wheeler et al. (25). were labelled by incubating cells with 1 g ml -1 lexa 594-Phalloidin (Invitrogen) for 2 hours at room temperature. were rinsed with TS and mounted using cytifluor (gar Scientific, UK) and TS. Optical sections (.5 m) and three-dimensional projections of specimens were obtained by the Leica TS NT confocal microscope. 4X objective was used for imaging. ll images were recorded using a stepper motor to make Z-series. Measurement of pollen tube length and diameter In order to determine if pollen tube growth was affected by.5% MSO or 5 µm Noc, pollen grains were allowed to germinate in K medium for one hour and then.5% MSO or 5 µm Noc was added to the culture. of Noc, MSO and control tubes (grown in K only) were taken after 15 and 45 min and cells were fixed as reported in the immunolabelling section. Images were captured using an xiovert 2 M microscope (Zeiss, Oberkochen, Germany) equipped with a 2X objective. Pollen tube lengths were measured by ImageJ software (National Institute of Health) at each time. To measure pollen tube diameters, cells grown for 45 min in the presence of.5% MSO or 5 M Noc were fixed as reported in the immunolabelling section. Medial plane images were collected by xiovert 2 M microscope, using a 63X objective. iameters were measured by ImageJ software (National Institute of Health). Statistical analysis of pollen tube lengths and diameters was performed by Excel software (Student s t-test). The frequency distribution of pollen tube diameters was analysed by IM SPSS 19 software. Vacuole labelling Tobacco pollen tubes grown in liquid medium were tranferred on polylisine-coated coverslips as reported in Methods. Vacuoles were stained with 3 M carboxy-(5(and-6-)carboxy-2,7 - dichlorofluorescein diacetate) (arboxy-f) (Invitrogen, US). Pollen tubes were observed before addition of drugs and at different times after incubation with MSO, Noc or Oryzalin. Vacuoles were imaged by 63X oil immersion (N 1.4) objective (Leica Microsystems, GmbH, Wetzlar, Germany) using the Leica TS SP2. arboxy-f was excited using the rgon 488 nm laser line and FM4-64 fluorescence was imaged between 55 and 54 nm. The distance of vacuoles from the apical PM was measured by ImageJ software (Schneider et al., 212) and analysed by (Student s t-test) by the program Excel.