Method S1
Plant genotyping
Homozygous individuals were identified by PCR genotyping using oligonucleotide primers
listed in Table S2. Primer combinations for the genotyping of the T-DNA insertion lines were
as follows: SALK_058170 (T-DNA allele 488, 3116; WT allele 2977, 3116), SALK_136917 (TDNA allele 488, 2977; WT allele 2977, 3116), GK787_E04 (T-DNA allele 3349, 448; WT
allele 3348, 3349), SAIL_31_D07 (T-DNA allele 2982, 1197; WT allele 2982, 2983). The
location of the T-DNA insertions were determined by left border sequencing.
Plasmid construction
Promoter or gene coding sequences were amplified by PCR from genomic DNA or cDNA
from pollen using Phusion DNA polymerase (Finnzymes) introducing new restriction sites at
the 5’- and 3’-termini (Table S2). PCR products were cloned into pJET1.2 (Fermentas) and
fully sequenced. RBOHH and RBOHJ coding sequences were introduced into a modified
pXNS vector (Cao et al. 2010) where the StrepII tag was replaced by a mCherry coding
sequence and the Pro35S was substituted with the pollen specific ProLAT52 promoter (Twell
et al. 1991). The RBOHH complementation construct is also based on the pXNS backbone
with an N-terminal EYFP tag and the upstream intergenic region of RBOHH (ProRBOHH675)
replacing the StrepII tag and Pro35S, respectively.
Plant transformation
Transgenic A.thaliana lines were generated by floral dip (Clough and Bent 1998) using the
Agrobacterium tumefaciens strain GV3101::pmp90RK carrying various pXNS constructs.
Complementation lines (CL) were generated with ProRBOHH675bp:YFP-RBOHH in the
rbohh-1
background.
The
ProLAT52:mCherry-RBOHH,
ProLat52:mCherry-RBOHJ
constructs were introduced into Col-0. Single insertion lines were selected by segregation
analysis of the BASTA resistance gene in the T2 generation.
Expression analysis by RT-PCR
RNA from root, shoot and leaf tissues was isolated using the TRIsure reagent (Bioline)
following the manufacturers´ instructions. Pollen RNA was isolated by collecting
approximately 100 freshly opened flowers in a microcentrifuge tube. Flowers were vortexed
in H2O and then briefly centrifuged (10 s at 5000 rpm) to release and collect the pollen. The
resulting pollen pellet was resuspended in 1 ml RNA ProTM solution (MP Biomedicals) and
transferred into a new tube containing lysing matrix B (MP Biomedicals). Pollen was
homogenized in a Retsch mixer mill for 10 min at 30 Hz. Following homogenization RNA
extraction was performed as described in the RNAproTM (MP Biomedical) instructions. cDNA
was synthesized using MMLV reverse transcriptase (Promega) and oligo-dT-primer. An
ubiquitin associated gene (At5g12120) was selected as internal control, since it is expressed
evenly in all tissues (Boavida et al. 2011). Primers used to detect RBOHH and RBOHJ
transcripts are listed in Table S2.
Aniline blue staining
Col-0 pistils were hand-pollinated with Col-0 or rbohh-1 rbohj-2 pollen and collected after 16
h. Pistils were incubated in fixative (acetic acid/EtOH; 1:3) for 2 h followed by treatment with
an EtOH series (70 %, 50%, 30% and ddH2O). The tissue was softened overnight in 8M
NaOH. After one washing step with ddH2O, pistils were treated with decolorized aniline blue
solution (0.1% [w/v] Aniline Blue; 108 mM K3PO4, pH 11, 2% [v/v] glycerol). Aniline Blue
stained pollen tubes were visualized by confocal microscopy using a 10 x /0.3NA objective
lens and following excitation emission parameters: Ex. 405 nm/Em. 500 nm - 530 nm.
References
Boavida, L.C., Borges, F., Becker, J.D. and Feijo, J.A. (2011) Whole genome analysis of
gene expression reveals coordinated activation of signaling and metabolic pathways
during pollen-pistil interactions in Arabidopsis. Plant Physiol, 155, 2066-2080.
Cao, F.Q., Werner, A.K., Dahncke, K., Romeis, T., Liu, L.H. and Witte, C.P. (2010)
Identification and characterization of proteins involved in rice urea and arginine
catabolism. Plant Physiol, 154, 98-108.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J, 16, 735-743.
Twell, D., Yamaguchi, J., Wing, R.A., Ushiba, J. and McCormick, S. (1991) Promoter
analysis of genes that are coordinately expressed during pollen development reveals
pollen-specific enhancer sequences and shared regulatory elements. Genes &
Development, 5, 496-507.