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Supplementary Information
The
Gibberellin
perception
system
evolved
to
regulate
a
pre-existing
GAMYB-mediated system during land plant evolution
Koichiro Aya1,*, Yuji Hiwatashi2,
3
, Mikiko Kojima4, Hitoshi Sakakibara4, Miyako
Ueguchi-Tanaka1, Mitsuyasu Hasebe2, 3, 5 & Makoto Matsuoka1
1
Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan.
2
National Institute for Basic Biology, Okazaki 444-8585, Japan.
3
Department of Basic Biology, School of Life Science, The Graduate University for Advanced
Studies, Okazaki 444-8585, Japan.
4
RIKEN Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan.
5
ERATO, Japan Science and Technology Agency, Okazaki 444-8585, Japan.
Supplementary Figures S1-S6
Supplementary Table S1
Supplementary Methods
Supplementary References
Supplementary Figure S1. Phylogenetic relationship between P. patens and S. moellendorffii
GAMYB homologs. (a) The phylogenic tree of the GAMYB homologs in representative green plants.
Black bar represents the cluster of GAMYB family. Scale bar, 0.1 substitution/site. (b) Protein sequence
alignment of P. patens and S. moellendorffii GAMYB homologs with flowering plant GAMYBs in three
conserved domains (BOX1-3).
Supplementary Figure S2. Conserved molecular function of SmCYP703C1 and PpCYP703B2. (a–d)
Outer wall structures of WT plants (a) and cyp703a3-1 rice plants transformed with the vector (b),
SmCYP703C1 (c) and PpCYP703B2 (d) (n>9). Ex, Exine; Scale bars, 1 μm.
Supplementary Figure S3. Rescue of OsGAMYB-mediated gene expression by SmGAMYB and
PpGAMYBs expression. (a, b) Scatter plot analysis to compare the expression pattern of
OsGAMYB-regulated genes by OsGAMYB and SmGAMYB expression (a) and OsGAMYB and
PpGAMYB2 expression (b) in the rice gamyb-2 background. Axes show the log2 value of the ratios of
signal intensities observed in the indicated transgenic plants relative to those in gamyb-2 plants. Each spot
represents one gene. (c) Change in the level of CYP703A3 mRNA upon expression of GAMYB homologs
in the rice gamyb-2 background. Data represent mean values ± s.d. (n=3).
Supplementary Figure S4. Effect of PpGAMYBs knockout on protonemal and gametophore
development in P. patens. (a, b) Morphology of a protonemal colony (a) and a gametophore (b) in WT
(left), PpGAMYB1-KO (middle), and PpGAMYB2-KO (right) P. patens. Scale bars, 1 mm. (c) Growth
rates of protonemal colonies in WT (blue), PpGAMYB1-KO (red), and PpGAMYB2-KO (green) P. patens.
Data represent mean values ± s.d. (n=9). The diameter on each gametophore was measured after the
cultivation on the BCDAT medium for indicated weeks.
Supplementary Figure S5. Expression pattern of PpGAMYBs and PpCYP703Bs in P. patens
sporangium. (a–d) In situ hybridization of PpGAMYB1 (a), PpGAMYB2 (c), PpCYP703B1 (b), and
PpCYP703B2 (d) with the antisense probe (left) or sense probe (right). Insets show close-up views. Sp,
Spore; Ss, Spore sac cell; Scale bars, 100 μm.
Supplementary Figure S6. Competitive gel shift assay using recombinant PpGAMYB2
protein. (a) Schematic representation of the 5’-flanking region of PpCYP703B2. The filled
circles show putative 8-bp GAMYB-binding-like motifs (TAACN[A/G/C]N[A/G/C]) with a
TAAC core sequence. Numbers at the bottom of the circles indicate the distance in base pairs
from the translational initiation site. Fragments, P1-4, were used as competitors for gel shift
assay in b. (b, c) Competition experiments were performed using increasing molar amounts
(X25, X50, X100) of the indicated unlabelled fragment. The competition activity was assesed
against the interaction between the DNA-binding (DB) domain of PpGAMYB2 and the
32P-labeled RAmy1A probe. The sequences of the oligonucleotides of PpCYP703B2 used in c
are shown. The putative binding site is underlined, and the mutations are indicated in lowercase
letters and the broken line.
Supplementary Table S1. Primer sequences.
Under-lines indicate the enzyme site used for cloning into the indicated vector.
Supplementary Methods
In silico screening and phylogenetic analysis.
The amino acid sequence of rice GAMYB was used as a query to screen S.
moellendorffii and P. patens genomic data of phytozome (http://www.phytozome.net/) by
TBLASTN, and candidate genes were selected for the phylogeneic analyses. For GAMYB genes,
candidate sequences were also manually checked and selected, based on the presence of
conserved amino acids important for their function and the miRNA159 target sequence18, 20.
After candidate sequences were obtained, the deduced full-length coding region for each gene
was PCR-amplified using cDNA of S. moellendorffii or P. patens as the template. Exon and
intron regions were confirmed by sequencing the PCR products. Multiple sequence alignments
and phylogenic tree were performed using Clustal X 2.0 and MEGA 4.0 with the
neighbor-joining method, JTT matrix, and complete deletion of gaps42, 43.
Microarray analysis.
Agilent 44K rice oligoarrays (G2519F, Agilent Technologies), which contain 44,000 features
with a 60-mer oligonucleotide, were used to generate two-color oligoarrays hybridized with rice
gamyb-2 mutant as a common reference sample. Each microarray experiment was performed
twice using RNA samples that had been isolated independently (i.e., two biological replicates),
and included a dye-swapping test as a technical replicate for each biological replicate. Labelling
and hybridization were performed according to the manufacturer's instruction manual. The
normalized signal ratios were normalized using the vsn method of R-Bioconductor44. To
identify differentially expressed genes, the values of log2 normalized signal ratio in each
experiment were statistically analyzed using a linear statistical model as described in the Limma
package of R-Bioconductor45. Using the P value and FDR calculated from an empirical Bayes
method of this package, 1322 OsGAMYB-regulated genes were identified with statistically
significant changes in expression between the anthers of OsGAMYB transgenic plants in an
gamyb-2 background and of gamyb-2 plants (at a P<0.001 and FDR<0.05). The expression
pattern of these 1322 OsGAMYB-regulated genes in the anthers of the OsGAMYB transgenic
plants was compared with those of SmGAMYB or PpGAMYB2 transgenic plants. Data are
available at Gene Expression Omnibus, accession code: GSE32652. All microarray data is
shown in Supplementary Data 1.
Gel shift assay.
To produce recombinant PpGAMYB2 protein, the cDNA fragment encoding the DNA-binding
domain of PpGAMYB2 (amino acids 1 to 365) was amplified from cDNA of P. patens, and was
then inserted into BamHI-SalI site of the pET-32a expression vector (Novagen) and expressed in
Rossetta-gami Escherichia coli cells (Takara Bio). To produce the DNA fragment of
PpCYP703B2 and RAmy1A, the 5’-flanking sequence of PpCYP703B2 and RAmy1A was
amplified from genomic DNA of P. patens or rice using the corresponding primers, and was
cloned into the pCR4 Blunt-TOPO vector. These resulting plasmids were utilized for the
production of the labeled fragments or the competitor fragments by the PCR amplification. The
amplified RAmy1A promoter fragment, digested with EcoRI, was labeled with 32P-dATP using
the Klenow fragment and purified on the NICK columns (GE Healthcare). The DNA binding
reactions were performed at 4 ℃ for 30 min in 10 mM Tris (pH7.5), 50mM NaCl, 1mM EDTA,
500 µM DTT, 10% glycerol, 0.05% tween-20, and 50mg/L poly(dI-dC)·poly(dI-dC) (Sigma),
and subjected to EMSA with 8% polyacrylamide gels in 0.25 × TBE buffer. For the competition
experiments, a 25, 50, and 100-fold molar excess of cold double-strand probe was added to the
reaction mixture. The primer sequences used in this study are listed in Supplementary Table S1.
Supplementary References
42. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948
(2007).
43. Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599 (2007).
44. Huber, W. et al. Variance stabilization applied to microarray data calibration and to the
quantification of differential expression. Bioinformatics 18, S96–S104 (2002).
45. Smyth, G. K. Linear models and empirical bayes methods for assessing differential
expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article 3 (2004).