supplementary materials for · from poo2-msl8 and poo2-msl8-yfp and stored at -80 °c at...
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www.sciencemag.org/content/350/6259/438/suppl/DC1
Supplementary Materials for
Mechanosensitive channel MSL8 regulates osmotic forces during pollen hydration and germination
Eric S. Hamilton, Gregory S. Jensen, Grigory Maksaev, Andrew Katims, Ashley M. Sherp, Elizabeth S. Haswell*
*Corresponding author. E-mail: [email protected]
Published 23 October 2015, Science 350, 438 (2015) DOI: 10.1126/science.aac6014
This PDF file includes:
Materials and Methods Figs. S1 to S8 Table S1 References
2
Materials and Methods
Plant material and growth conditions.
Plants were grown on soil under 24 hours of light at 21 °C. msl8-4 (DsLoxN101568 and
DsLoxN101751, in the Ler background) and msl8-1 (SALK_004922, in the Col-0 background) were
obtained from the Arabidopsis Biological Resource Center.
Microscopy.
Brightfield and epifluorescent images were acquired on an Olympus BX-61 microscope using an
Olympus DP71 digital camera, DP Controller software, and filter sets for GFP (470/40 nm excitation,
525/50 nm emission), YFP (500/20 nm excitation, 535/30 nm emission), and/or dsRED (545/30 nm
excitation, 620/60 nm emission). Confocal images of pollen and Xenopus oocytes were acquired on the
same microscope with FV10-ASW Olympus software using the DAPI (488 nm excitation, 430-470 nm
bandpass filter), GFP (488 nm excitation, 505-605 nm bandpass filter), YFP (515 nm excitation, 535-
565 nm bandpass filter), and/or mCherry (543 nm excitation, 560-660 nm bandpass filter) channels.
For environmental scanning electron microscopy, mature pollen grains were tapped from anthers onto a
piece of tape on a sample mount and imaged directly using a Hitachi TM-1000 tabletop SEM at 15 kV.
Images of whole plants were taken with a 10-megapixel cell phone camera.
Reverse transcriptase-polymerase chain reaction.
cDNAs were generated using an oligo(dT)20 primer and M-MLV Reverse Transcriptase
(Promega) from template RNA extracted from the indicated tissues with either TRIzol Reagent
(Invitrogen) or the RNeasy Mini RNA extraction kit (Qiagen). ACTIN, MSL8, and MSL8-YFP
transcripts were amplified with the primers indicated in Supplementary Table 1 using 20, 27, and 37
cycles, respectively. A mixture of three reverse ACT primers was used to target three ACTIN paralogs.
The leaf, root, and flower ACTIN and MSL8 genes were amplified in 30 cycles. Quantitative RT-PCR
was performed as previously described (27). Forward and reverse primers (described in Supplemental
Table 1) were added to a cocktail containing 1X SYBR Green PCR Master Mix (Applied Biosciences)
and 0.5 µL cDNA to make a final 25 µL reaction. After amplification in the StepOnePlus real-time
PCR system, the data was analyzed using StepOne software (Applied Biosciences).
DAPI staining.
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Anthers were dissected from flowers at the indicated stages and placed into 25 µl of pollen
isolation buffer (100 mM NaPO4 pH 7.5, 1 mM EDTA, 0.1% (v/v) Triton X-100) containing 3 µg/ml
4', 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) on a microscope slide. A coverslip was pressed
against the dissected anther to release pollen and the samples were imaged after 5 minutes.
Subcellular localization of MSL8.
MSL8-YFP-expressing plants were crossed to CPK34-mCherry-, ER-mCherry, or vacuole-mCherry-
expressing plants and their progeny selected for resistance to both kanamycin and Bialaphos (Basta).
MSL8-YFP-expressing plants were transformed with LAT52pGolgi-mCherry and LAT52pVacuole-
mCherry constructs via agrobacterium and the T1 generation selected for resistance to both kanamycin
and Basta. Pollen grains from stage 13/stage 14 flowers (26) were released into 25-30 µl drops of
water or pollen germination media as below on double ring cytology slides (Fischer Scientific) by
dipping inverted flowers. Either immediately or after overnight germination as described below, a
coverslip was added and pollen imaged by confocal microscopy, scanning sequentially in the GFP,
mCherry and pseudo-DIC channels.
Electrophysiology.
All the traces presented in this report were obtained from inside-out (excised) patches.
Complementary RNA production, oocyte retrieval and injection, and single channel patch clamp
electrophysiology were performed as described in (28). Capped MSL8 cRNA was transcribed in vitro
from pOO2-MSL8 and pOO2-MSL8-YFP and stored at -80 °C at approximately 1000 ng/µl until use.
Xenopus laevis oocytes (Dumont stage V or VI) were collected, injected with 50 µl pOO2-MSL8
cRNA, pOO2-MSL8-YFP cRNA or water, and patched after one week. The buffers used were:
modified complete ND96 (96 mM NaCl, 2 mM KCl, 2 mM CaCl2, 10 mM MgCl2, 5 mM Hepes, pH
7.38) and 60 mM MgCl2 with 5 mM Hepes. In some experiments, 10 µM Ruthenium Red (Sigma) was
added to both pipette and bath buffers, or 450 mM sorbitol was added to the bath solution, or 100 mM
TEA-Cl (Sigma) was used instead of 96 mM NaCl in ND96, as indicated in figure legends.
Pollen protoplasts were isolated by treating Col-0 pollen grains in buffer (1 mM KNO3, 0.2 mM
KH2PO4, 1 mM MgSO4, 1 µM KI, 0.1 µM CuSO4, 5 mM CaCl2, 5mM MES, 500 mM glucose, 500
mM sorbitol, Tris added to pH 5.8) with 2 % (w/v) cellulase (Sigma), 2% (w/v) pectinase (Sigma),
0.4% (w/v) BSA added. The suspension was filtered to remove debris prior to patching experiments. In
pollen protoplasts were patched with pipette bubble number ~4.5 - 5, transmembrane potential -60 mV.
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Buffers were 60 mM MgCl2 (pipette) and 60 mM MgCl2, 450 mM Sorbitol (bath) buffers buffered with
HEPES and pH adjusted with TEA-OH. 100 mM TEA-Cl was added to both buffers where indicated.
Experiments in asymmetric buffers (100/300 mM NaCl) utilized Rainin Minipulse3 peristaltic
pumps. In all measurements with asymmetric buffers liquid junction potentials were corrected after the
patch was broken. Electrode potential drift was tested before the experiments and was less than 0.1 mV
per 10 min. The reversal potential of MSL8 was approximately -18 mV, which results in a permeability
ratio (PCl : PNa) of 6.3 for MSL8 while the theoretical reversal potential of Cl- ion, derived from the
Nernst equation under a three-fold gradient of ion concentration, is -28 mV. According to the
Goldman-Hodgkin-Katz equation, this results in a permeability ratio (PCl : PNa) of 6.3 for MSL8.
Calculation of transmission ratios.
The transmission efficiency of the msl8-4 allele was determined by reciprocally crossing msl8-4
heterozygotes to Ler as either the male or female parent. The progeny were genotyped by PCR as
described below and the number of wild type and msl8-4 heterozygous plants determined. The
transmission ratio of the LAT52pMSL8-YFP transgene was determined by selecting the T2 generation
with Basta on soil and counting the number of resistant and sensitive progeny.
Genotyping.
DNA was extracted by grinding tissue in extraction buffer (200 mM Tris-HCl pH 7.5, 250 mM
NaCl, 250 mM EDTA, 0.5% SDS) and precipitating with isopropanol. Wild type MSL8, msl8-1, and
msl8-4 alleles were genotyped by PCR using the primers in Supplementary Table 1 and 30-31 cycles of
amplification.
In vitro pollen hydration.
Pollen from mature (stage 13-14) flowers was released into 25-30 µl drops of PI/FDA stain (1
µg/ml fluorescein diacetate (FDA, Sigma-Aldrich) and 0.5 µg/ml propidium iodide (PI, Sigma-Aldrich)
in deionized water) on double ring cytology slides by dipping inverted flowers. Slides were incubated
upside down in a humid chamber at room temperature. After the indicated time points, a coverslip was
added and FDA and PI fluorescence was imaged in the GFP and dsRED epifluorescent channels,
respectively. FDA stains live cells; PI stains the edge of live cells and enters compromised cells. In
some experiments, pollen was hydrated in the indicated percent (w/v) polyethylene glycol (PEG,
average molecular weight 3350g/mol, Sigma-Aldrich) solution. In some experiments, pollen was
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hydrated in the indicated water or PEG solution without stain to which an equal volume of 2X PI/FDA
stain prepared in the appropriate solution was added prior to imaging.
In vitro pollen germination.
Germination was performed essentially as in (29). Wild type and pollen was released into liquid
germination media (10% sucrose, 0.01% boric acid, 5 mM CaCl2, 5 mM KCl, 1 mM MgSO4, pH 7.5)
and incubated in a humid chamber at room temperature. After four hours, 15 µl of 0.01% decolorized
aniline blue (Sigma-Aldrich) solution and a coverslip were added. Aniline blue signal was detected in
the DAPI channel. Each imaged pollen grain was categorized as ungerminated; ungerminated and
burst; germinated; or germinated and burst with aniline blue staining of callose used as a marker of
germination.
Wild type and LAT52pMSL8-YFP pollen was incubated overnight on agarose pads (germination
media solidified with 1.5% agarose (Bioexpress)) by brushing 3-4 inverted flowers and incubated in
humid chambers at room temperature. For in vivo pollen germination, gMSL8-GFP pollen was applied
to the stigma of a gMSL8-GFP plant. After pollen germinated on the benchtop, the stigma was
dissected and mounted in water on a slide for confocal imaging in the GFP channel.
In vivo pollen hydration.
Pistils from male sterile 1 (ms1-1, Ler ecotype) flowers were excised and embedded vertically in
1% agarose on a slide such that the stigma protruded above the gel surface. Pollen was applied to the
stigma from an anther dissected from mature (stage 13-14) flowers under a dissecting microscope. The
slide was quickly moved to an Olympus BX-61 microscope where hydration of an individual pollen
grain attached to a stigma cell was then tracked with an Olympus DP71 digital camera and DP
Controller software under a 20X objective. Brightfield images were captured every 60 seconds for 20
minutes and the equatorial diameter of each pollen grain at each time point measured in ImageJ.
Subcloning.
To make gMSL8-GFP, the MSL8 gene (including all genomic sequence from 588 bases upstream
of the MSL8 start codon to 731 bases after the MSL8 stop codon) was amplified from genomic Col-0
DNA using gene-specific primers [1686 + 1687] and cloned into pENTR/D-TOPO (Life Technologies)
to make pENTR/gMSL8. An EcoRI site was introduced between the MSL8 sequence and the 3’UTR by
site-directed mutagenesis and eGFP was inserted to make pENTR/gMSL8-GFP. pBGW/gMSL8 and
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pBGW/gMSL8-GFP were generated by LR recombination with LR Clonase II enzyme (Life
Technologies) into pBGW (30) according to the manufacturer’s specifications.
Three overlapping fragments of the MSL8 cDNA were amplified from a Col-0 flower cDNA
library using primer pairs: [627 + 1772 (5’ end)], [765 + 1770 (middle)], [767 + 670 (3’ end)] and
cloned into pGEM T-Easy (Promega). The full-length 2727 bp cDNA was then generated by fusion
PCR using the three templates and either [668 + 670] or [668 + 671] primer pairs and cloned into
pENTR/D-TOPO to make pENTR/MSL8 or pENTR/MSL8(no stop), respectively. The sequence of this
cDNA was consistent with the splicing information obtained from RNA-Seq in (13). pENTR/MSL8
and pENTR/MSL8(no stop) were recombined with pGWOO2 and pGWOO2-YFP-HA (31) to create
pOO2-MSL8 and pOO2-MSL8-YFP, respectively.
To make LAT52pMSL8-YFP, pENTR-/MSL8-YFP was created by amplifying the MSL8-YFP
fragment from pOO2-MSL8-YFP using primers [668 + 1753] and recombined with pKLAT52GW7
and pB7WGLAT52 vectors (32). The coding sequences of G-rk, er-rk and v-rk from (Nelson et al.,
2007) were cloned into D-TOPO/pENTR to make pENTR/Golgi-mCherry, pENTR/ER-mCherry and
pENTR/Vacuole-mCherry. To make pENTR/CPK34-mCherry, PCR was used to add NotI and XhoI
sites to the coding sequence of CPK34 with primers 2352 and 2353 to ligate into pENTR/Golgi-
mCherry to make pENTR/CPK34-mCherry. To make LAT52pCPK34-mCherry, LAT52pER-mCherry
and LAT52pVacuole-mCherry, pENTR/CPK34-mCherry, pENTR/ER-mCherry and pENTR/Vacuole-
mCherry were recombined into the pKLAT52GW7 binary vector. All constructs were transformed into
the Agrobacterium tumefaciens GV3101 strain and into Arabidopsis thaliana using the floral dip
method.
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8
Fig. S1 MSL8 alignment, topology, and expression in pollen.
(A) Amino acid sequence conservation between the conserved domain of selected MscS family
members, including MscS from Escherichia coli and MscS-Like (MSL)7, MSL8 and MSL10 from
Arabidopsis thaliana. Identical residues are indicated by an asterisk. The upper and lower portions of
the pore-lining helix of MscS are indicated as TM3a and TM3b, respectively. (B) Known and predicted
topologies of MscS and MSL8, respectively. Grey bar indicates the plasma membrane; each circle is an
amino acid. Yellow color indicates the conserved MscS domain, dark grey are known (MscS) or
predicted (MSL8, aramemnon.org) transmembrane helices. (C) Reverse transcription-PCR performed
on mRNA isolated from flowers (F), leaves (L) or roots (R) from 2 week-old seedlings, or on genomic
DNA (g). (D) RNA sequencing reads for all seven plasma membrane-localized MSL genes in A.
thaliana mined from a high throughput sequencing of pollen and seedling transcriptomes (13). AGI,
Arabidopsis Genome Initiative; RPM, reads per million; RPKM, reads per kilobase million.
9
Fig. S2. Male-specific expression is observed in MscS homologs from monocots and dicots.
(A) Table of selected MscS homologs predicted to localize to the plasma membrane from various plant
species and their expression pattern based on the indicated databases. (B) Phylogenetic relationship
between the homologs listed in (A). Full-length proteins were aligned in Clustal 2 with a gap-opening
penalty of 3.0 and a gap extension penalty of 1.8. The evolutionary history was inferred using the
Neighbor-Joining method using MEGA6 software. The reliability of the tree was determined via
bootstrapping (n = 100 replicates). Genes where evidence for male-specific (anther, pollen, or male
floral organ) expression has been collected are indicated in blue.
10
11
Fig. S3. MSL8-YFP localizes to the plasma membrane and does not co-localize with and
endoplasmic reticulum marker.
Quantification of co-localization of MSL8-YFP and CPK34-mCherry (a marker for the plasma
membrane) or endoplasmic reticulum-mCherry expressed under control of the LAT52 promoter. (A-C)
Confocal images of MSL8-YFP (pseudocolored green) and CPK34-mCherry (pseudocolored red). (A)
both channels; (B) red channel (C) green channel. (D-F) Confocal images of MSL8-YFP
(pseudocolored green) and endoplasmic reticulum-mCherry (pseudocolored red). (D) both channels;
(E) red channel (F) green channel. Regions of interest (ROIs) were selected in the red channels,
indicated by numbers (B, E). (G-L) details of ROIs chosen for Pearson’s correlation coefficient
analysis with plasma membrane (G-J) and endoplasmic reticulum (K, L) markers. Co-localization was
quantified using the scatterplot method and calculating Pearson’s correlation coefficients (presented in
(M)) with the Coloc 2 plug-in for Fiji (http://fiji.sc/Coloc2).
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Figure S4. Confocal images of pollen expressing MSL8-YFP with Golgi- or vacuole-mCherry
membrane markers.
(A-C) Co-expression of MSL8-YFP (pseudocolored green) with Golgi-mCherry (pseudocolored red).
(A) green channel (B) red channel (C) green and red channels merged. (D-F) Co-expression of MSL8-
YFP (pseudocolored green) with vacuole-mCherry (pseudocolored red). (D) green channel. (E) red
channel. (F) green and red channels merged. Scale bars are 10 µm.
13
Fig. S5. MSL8 electrophysiology in oocytes
(A, B) Representative confocal images of the MSL8-YFP cRNA injected oocyte periphery (A, 20x
lens, fluorescence and bright field merged) and a fraction of the flattened oocyte membrane (B, 100x
lens). The images were taken 7 days after RNA injection. (C) Comparison of unitary conductance of
MSL8 (squares) and MSL8-YFP (diamonds) expressed in oocytes in ND96, and MSL8 expressed in 60
mM MgCl2 (triangles) buffers. Excised inside-out patches from oocyte membrane, pipette BN 4.5 -5.
In ND96 MSL8 unitary conductance was 58 pS under negative and 40 pS under positive membrane
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potentials (D) Representative trace of threshold tension for MscS. (E) Representative trace of threshold
tension for MSL8. The threshold tension for opening was approximately -160 mm Hg while the last
channel closed at -70 mm Hg. Note that MSL8 exhibited asymmetric opening and closing kinetics.
15
Fig S6. Sample traces and I/V curves from pharmacological characterizations of MSL8 in oocytes
and in pollen protoplasts.
(A) Activation of MSL8-YFP expressed in oocytes by tension in presence of symmetric 10 µM
Ruthenium Red in ND96 buffer, -40 mV membrane potential, pipette “bubble number” (BN) 5. (B) At
this concentration, Ruthenium Red did not affect unitary conductance of MSL8-YFP. Unitary
conductance was found to be 60 pS in 0 mV to -80 mV membrane potential range and 44 pS between 0
mV and 80 mV membrane (squares). The I-V relationship for MSL8-YFP in the absence of Ruthenium
Red is labeled as open circles. (C) Activation of MSL8-YFP expressed in oocytes by tension in
symmetric 100 mM TEA-Cl buffer -60 mV membrane potential, pipette BN 4.5. (D) Activation of
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MSL8-YFP expressed in oocytes by tension in asymmetric TEA-Cl buffer (100 mM TEA-Cl in pipette
buffer, 100 mM TEA-Cl + 450 mM Sorbitol in bath), -40 mV membrane, pipette BN 4.5. Multiple
conductive sub-states were observed, and are labeled S1-S2. Complete openings are labeled O1-O3.
(E) Adding 450 mM Sorbitol to the bath solution slightly decreased the unitary conductance of MSL8-
YFP expressed in oocytes. Under symmetric conditions, the unitary conductance measured 57 pS under
negative membrane potentials and 49pS under positive membrane potentials (100 mM TEA-Cl buffer,
square symbols). Under asymmetric conditions (100 mM TEA-Cl in a pipette and 100 mM TEA-Cl
with 450 mM Sorbitol in bath, diamonds), the unitary conductance of MSL8 was 42pS under negative
membrane potentials and 39pS under positive membrane potentials. (F) Representative trace from
excised inside-out patch from Columbia pollen protoplasts in 100 mM TEA-Cl (pipette) and 100 mM
TEA-Cl supplied with 450 mM Sorbitol (bath) buffers. Membrane potential is -40mV, pipette BN 4.
The differences between closed (C) and open (O) states correspond to the single channel conductance
of MSL8 expressed in oocytes and measured under the same conditions. The inset trace also suggests
the presence of multiple conducting sub-states.
17 Fig.
18
S7. Structure of msl8 mutant alleles and molecular complementation of the hydration viability
phenotype with gMSL8-GFP
(A) Diagram of msl8 insertion alleles. The msl8-1 insertion is 47 bp upstream of the MSL8 start codon;
the msl8-4 insertion is 147 bp downstream of the MSL8 start codon. The location of the GFP insertion
in the gMSL8-GFP construct is also indicated. (B) Quantitative RT-PCR of MSL8 transcript levels,
presented relative to ACTIN transcript levels, in the msl8-1 and msl8-4 mutants. Two technical
replicates of three biological replicates are presented. Error bars represent standard error. (C, D)
Complementation of the hydration viability phenotype in msl8-1 (C) and msl8-4 (D) mutant
backgrounds by the gMSL8-GFP construct, which contains the entire genomic sequence encoding
MSL8, including promoter and 3’UTR. Pollen was released into distilled water and incubated for 2
hours before staining with vital stains FDA and PI to determine number of live pollen n = 70-180
pollen grains per line. All lines presented are homozygous for the gMSL8-GFP transgene and exhibited
75% Bialophos (Basta) resistance in the T2 generation, indicating a single T-DNA insertion event.
Homozygous T2 individuals were identified as those with close to 100% GFP signal in pollen grains
developing inside their stamen (representative images shown to the right of each chart) and assays were
performed on pollen isolated from these T2 plants. These data provide strong evidence that the defect
in viability after hydration in msl8-1 and msl8-4 mutant pollen can be attributed to lesions in the MSL8
gene. (E) Diameter of msl8-4 and Ler pollen grains during in vivo hydration on an excised stigma. No
significant (p > 0.05 by Student’s t-test) differences between wild type and msl8-4 pollen were
observed. N > 37 per genotype. Error bars are standard error.
19
Fig. S8. LAT52pMSL8-YFP-expressing plants are impaired in male fertility, but their pollen
grains survive hypoosmotic stress similar to wild type
(A) Frequency of homozygote recovery in the T2 generation for LAT52pMSL8-YFP lines 2-1, 11, 27
and 11. Homozygotes were identified following selection with Basta for the transgene by screening
individual plants for close to 100% YFP signal in pollen grains released into water. (B) Reciprocal
crosses between a heterozygote for LAT52pMSL8-YFP line 2-1 (a strong expresser) and the wild type
were performed, and the resulting F1 progeny selected for transgene-encoded Basta resistance. The
defect in transmission is only observed when the transgene is provided by the male parent. (C)
Quantification of results in (B). (D) Percent viability of wild type and LAT52pMSL8-YFP pollen in
distilled water and 20% PEG as determined by FDA and PI staining.
20
Table S1. Primers used in genotyping, quantitative PCR, and cloning reactions.
References 1. A. Anishkin, S. H. Loukin, J. Teng, C. Kung, Feeling the hidden mechanical forces in lipid
bilayer is an original sense. Proc. Natl. Acad. Sci. U.S.A. 111, 7898–7905 (2014). Medline doi:10.1073/pnas.1313364111
2. C. D. Pivetti, M. R. Yen, S. Miller, W. Busch, Y. H. Tseng, I. R. Booth, M. H. Saier Jr., Two families of mechanosensitive channel proteins. Microbiol. Mol. Biol. Rev. 67, 66–85 (2003). Medline doi:10.1128/MMBR.67.1.66-85.2003
3. E. S. Hamilton, A. M. Schlegel, E. S. Haswell, United in diversity: Mechanosensitive ion channels in plants. Annu. Rev. Plant Biol. 66, 113–137 (2015). Medline doi:10.1146/annurev-arplant-043014-114700
4. I. R. Booth, P. Blount, The MscS and MscL families of mechanosensitive channels act as microbial emergency release valves. J. Bacteriol. 194, 4802–4809 (2012). Medline doi:10.1128/JB.00576-12
5. E. S. Haswell, R. Peyronnet, H. Barbier-Brygoo, E. M. Meyerowitz, J. M. Frachisse, Two MscS homologs provide mechanosensitive channel activities in the Arabidopsis root. Curr. Biol. 18, 730–734 (2008). Medline doi:10.1016/j.cub.2008.04.039
6. N. Firon, M. Nepi, E. Pacini, Water status and associated processes mark critical stages in pollen development and functioning. Ann. Bot. 109, 1201–1214 (2012). Medline doi:10.1093/aob/mcs070
7. G. G. Franchi, B. Piotto, M. Nepi, C. C. Baskin, J. M. Baskin, E. Pacini, Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival. J. Exp. Bot. 62, 5267–5281 (2011). Medline doi:10.1093/jxb/err154
8. A. F. Edlund, R. Swanson, D. Preuss, Pollen and stigma structure and function: The role of diversity in pollination. Plant Cell 16 (suppl. 1), S84–S97 (2004). Medline doi:10.1105/tpc.015800
9. H. J. Wang, J. C. Huang, G. Y. Jauh, Pollen germination and tube growth. Adv. Bot. Res. 54, 1–52 (2010). doi:10.1016/S0065-2296(10)54001-1
10. L. Beauzamy, N. Nakayama, A. Boudaoud, Flowers under pressure: Ins and outs of turgor regulation in development. Ann. Bot. 114, 1517–1533 (2014). Medline doi:10.1093/aob/mcu187
11. J. Kroeger, A. Geitmann, The pollen tube paradigm revisited. Curr. Opin. Plant Biol. 15, 618–624 (2012). Medline doi:10.1016/j.pbi.2012.09.007
12. E. Michard, F. Alves, J. A. Feijó, The role of ion fluxes in polarized cell growth and morphogenesis: The pollen tube as an experimental paradigm. Int. J. Dev. Biol. 53, 1609–1622 (2009). Medline doi:10.1387/ijdb.072296em
13. J. A. Feijo, R. Mahlo, G. Obermeyer, Ion dynamics and its possible role during in vitro pollen germination and tube growth. Protoplasma 187, 155–167 (1995). doi:10.1007/BF01280244
14. R. Dutta, K. R. Robinson, Identification and characterization of stretch-activated ion channels in pollen protoplasts. Plant Physiol. 135, 1398–1406 (2004). Medline doi:10.1104/pp.104.041483
15. A. E. Loraine, S. McCormick, A. Estrada, K. Patel, P. Qin, RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing. Plant Physiol. 162, 1092–1109 (2013). Medline doi:10.1104/pp.112.211441
16. D. Twell, T. M. Klein, M. E. Fromm, S. McCormick, Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol. 91, 1270–1274 (1989). Medline doi:10.1104/pp.91.4.1270
17. C. Myers, S. M. Romanowsky, Y. D. Barron, S. Garg, C. L. Azuse, A. Curran, R. M. Davis, J. Hatton, A. C. Harmon, J. F. Harper, Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes. Plant J. 59, 528–539 (2009). Medline doi:10.1111/j.1365-313X.2009.03894.x
18. S. Frietsch, Y. F. Wang, C. Sladek, L. R. Poulsen, S. M. Romanowsky, J. I. Schroeder, J. F. Harper, A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen. Proc. Natl. Acad. Sci. U.S.A. 104, 14531–14536 (2007). Medline
19. M. Tunc-Ozdemir, C. Rato, E. Brown, S. Rogers, A. Mooneyham, S. Frietsch, C. T. Myers, L. R. Poulsen, R. Malhó, J. F. Harper, Cyclic nucleotide gated channels 7 and 8 are essential for male reproductive fertility. PLOS ONE 8, e55277 (2013). Medline doi:10.1371/journal.pone.0055277
20. G. Maksaev, E. S. Haswell, Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes. J. Gen. Physiol. 138, 641–649 (2011). Medline doi:10.1085/jgp.201110723
21. G. Maksaev, E. S. Haswell, Recent characterizations of MscS and its homologs provide insight into the basis of ion selectivity in mechanosensitive channels. Channels 7, 215–220 (2013). Medline doi:10.4161/chan.24505
22. R. Arunkumar, E. B. Josephs, R. J. Williamson, S. I. Wright, Pollen-specific, but not sperm-specific, genes show stronger purifying selection and higher rates of positive selection than sporophytic genes in Capsella grandiflora. Mol. Biol. Evol. 30, 2475–2486 (2013). Medline doi:10.1093/molbev/mst149
23. M. Bialecka-Fornal, H. J. Lee, R. Phillips, The rate of osmotic downshock determines the survival probability of bacterial mechanosensitive channel mutants. J. Bacteriol. 197, 231–237 (2015). Medline doi:10.1128/JB.02175-14
24. O. Hamant, Widespread mechanosensing controls the structure behind the architecture in plants. Curr. Opin. Plant Biol. 16, 654–660 (2013). Medline doi:10.1016/j.pbi.2013.06.006
25. C. J. Miller, L. A. Davidson, The interplay between cell signalling and mechanics in developmental processes. Nat. Rev. Genet. 14, 733–744 (2013). Medline doi:10.1038/nrg3513
26. M. E. Wilson, G. S. Jensen, E. S. Haswell, Two mechanosensitive channel homologs influence division ring placement in Arabidopsis chloroplasts. Plant Cell 23, 2939–2949 (2011). Medline doi:10.1105/tpc.111.088112
27. D. R. Smyth, J. L. Bowman, E. M. Meyerowitz, Early flower development in Arabidopsis. Plant Cell 2, 755–767 (1990). Medline doi:10.1105/tpc.2.8.755
28. G. Maksaev, E. S. Haswell, Expressing and characterizing mechanosensitive channels in Xenopus oocytes. Methods Mol. Biol. 1309, 151–169 (2015). Medline doi:10.1007/978-1-4939-2697-8_13
29. L. C. Boavida, S. McCormick, Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J. 52, 570–582 (2007). Medline doi:10.1111/j.1365-313X.2007.03248.x
30. M. Karimi, D. Inzé, M. Van Lijsebettens, P. Hilson, Gateway vectors for transformation of cereals. Trends Plant Sci. 18, 1–4 (2013). Medline doi:10.1016/j.tplants.2012.10.001
31. K. M. Veley, G. Maksaev, E. M. Frick, E. January, S. C. Kloepper, E. S. Haswell, Arabidopsis MSL10 has a regulated cell death signaling activity that is separable from its mechanosensitive ion channel activity. Plant Cell 26, 3115–3131 (2014). Medline doi:10.1105/tpc.114.128082
32. K. E. Francis, S. Y. Lam, B. D. Harrison, A. L. Bey, L. E. Berchowitz, G. P. Copenhaver, Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 104, 3913–3918 (2007). Medline doi:10.1073/pnas.0608936104