Biology Reference
In-Depth Information
of 10
-8
by TBLASTN [81]. For comparisons with sporophytic transcriptomes,
the highly standardized experiment conducted by Schmid and coworkers [28]
was chosen. Presence/absence calls calculated from the microarray analyses
were downloaded for selected tissues from the TAIR website [83]. A gene was
declared to be expressed in a tissue when a presence call was assigned to it in
at least two out of three replicates.
In Situ Hybridization
Inflorescences and emasculated pistils were paraplast embedded using the pro-
tocol of Kerk and colleagues [84] with minor modifications. Unique gene-
specific probes of about 200 to 300 base pairs were cloned into pDRIVE
(Qiagen, Basel, Switzerland) and used as templates for generating digoxy-
genin-UTP-labeled riboprobes by run-off transcription using T7 RNA poly-
merase, in accordance with the manufacturer's protocol (Roche Diagnostics,
Basel, Switzerland). In situ hybridization was performed on 8 to 10
µ
m semi-
thin paraffin sections, as described by Vielle-Calzada and coworkers [85] with
minor modifications.
Histochemical GUS Expression
Embryo sac expression of the GUS reporter gene (
β
-glucuronidase) in the pro-
moter-GUS lines and transposants was detected as described by Vielle-Calzada
and coworkers [86].
image processing
All of the images were recorded using a digital Magnafire camera (Optronics, Go-
leta, CA, USA), and they were edited for picture quality using Adobe Photoshop
version CS (Adobe Systems Inc., San Jose, CA, USA).
Abbreviations
BLAST, basic local alignment search tool; coa, coatlique mutant; dCHIP, DNA-
Chip Analyzer; FDR, false discovery rate; gcRMA, GC robust multi-array average;
MAS, MicroArray Suite (Affymetrix); RT-PCR, reverse transcription polymerase
chain reaction; spl, sporocyteless mutant; TAIR, The Arabidopsis Information
Resource.
