Signalling components of BABY BOOM-induced

somatic embryogenesis
Anneke Horstman, Mieke Weemen, Gerco Angenent, Kim Boutilier
Plant Research International, Wageningen University
PO Box 619, 6700 AP Wageningen, The Netherlands
Telephone: + 31 317 480 944
E-mail: anneke.horstman@wur.nl
BBM interacting partners
1. Boutilier, K., Offringa, R., Sharma, V.K., Kieft, H., Ouellet, T., Zhang, L., Hattori, J., Liu, C.,
Lammeren, A.A.M van, Miki, B.L.A., Custers, J.B.M., Lookeren Campagne, M.M. van (2002). Ectopic
expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. The Plant
Cell 14: 1737-1749
2. Passarinho, P.A., Ketelaar, M.J., Xing, M., Arkel, J. van, Maliepaard, C.A., Weemen, W.M.J., Joosen,
R.V.L., Lammers, M., Herdies, L., Boer, B. de, Geest, A.H.M. van der, Boutilier, K.A. (2008). BABY
BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways. Plant
Molecular Biology 68: 225-237
Table 1: Top ten candidate BABY BOOM target genes
BBM targets
5.66 0.21 4.14 0.06 Calcium-binding EF hand protein At2g34020 10
5.14 0.26 4.39 0.14 Leucine-rich receptor-like kinase, LRRII group At5g45780 9
5.57 0.54 4.58 0.15 RING H2 domain protein At3g54780 8
9.45 0.45 4.78 0.09 Actin-depolymerizing factor (ADF9) At4g34970 7
8.40 0.09 4.78 0.07 Tubby family protein (TLP8) At1g16070 6
- 4.79 0.42 Unknown protein At2g03830 5
6.02 0.48 4.89 0.04 NAC domain protein (ANAC094) At5g39820 4
5.99 0.27 4.94 0.22 Cytochrome P450 (CYP71B22) At3g26200 3
5.54 1.72 5.21 0.25 BTB-POZ domain protein, NPH3 family (NRL27) At5g48130 2
5.98 1.00 6.08 0.09 BABY BOOM (BBM) At517430 1
qRT-PCR Microarray (2Log ratio) Annotation AGI No. Rank
Microarray analysis was used in combination with a post-translationally regulated
BBM:GR protein and cycloheximide to identify target genes that are directly
activated by BBM in arabidopsis seedlings
2
. The ten highest upregulated genes
are shown in Table 1. None of the targets are functionally characterized, except
for BBM itself.
Introduction
Embryogenesis can be induced from somatic cells by stress- and/or growth
regulator treatments, a process named somatic embryogenesis (SE). Ectopic
expression of the Brassica napus AP2/ERF domain transcription factor BABY
BOOM (BBM) is sufficient to induce SE in several plant species in the absence of
any exogenous treatment
1
(Figure 1). To gain more insight into the BBM
signalling pathway, we are studying its
downstream gene targets and
interacting protein partners using ChIP-
seq and Split-YFP. In addition, we are
comparing the functions of the B. napus
and arabidopsis BBM proteins.
Figure 1: Arabidopsis 35S::BBM plants
forming somatic embryos
Figure 3: Detection of protein-protein interactions with Split-YFP in protoplasts
B. napus BBM versus A. thaliana BBM
The overall amino acid similarity between arabidopsis and Brassica BBM is 85%,
while the similarity in the region spanning the two AP2 domains is even higher
(99%). Furthermore, the intron-exon boundaries are perfectly conserved. However,
35S::BnBBM induces SE much more efficiently than 35S::AtBBM in the same vector
background (Table 2). We are
testing whether presence or
absence of specific BBM
regulatory elements in the two
genes accounts for this
difference.
Two BBM interacting proteins were identified in a yeast-two-hybrid screen:
PICKLE-RELATED1 (PKR1) and HOMEODOMAIN GLABROUS11 (HDG11).
PKR - CHD3 chromatin remodeler
- no mutant phenotype
- related to PICKLE (= repressor
of embryogenic traits)
HDG11 - transcription factor of the HD-
ZIP IV family
- involved in drought tolerance
and trichome branching
We are determining whether loss or gain of function mutants of PKR1 and/or
HDG11 influence BBMs ability to form somatic embryos.
Split-YFP
We confirmed these interactions in planta by using the split-YFP system in
protoplasts (Figure 3).
Literature
2 (0.5%) 400 35S::AtBBM
19 (22%) 81 35S::BnBBM
Embryogenic lines Number of T1 lines Construct
ChIP-seq
ChIP-seq (chromatin immunoprecipitation with subsequent sequencing)
experiments are now being performed in somatic embryo cultures (Figure 2a) to
validate BBM targets and identify those that are expressed during early somatic
embryo development. A BBM:YFP fusion protein and a YFP antibody is being
used to purify the BBM:YFP-DNA complexes (Figure 2b-d).
Figure 2: Material for ChIP-seq. Somatic embryo tissue culture induced by 2,4-D (A),
pBBM::BBM:YFP expression in root (B), embryo (C) and somatic embryo sections (D).
Protein blot of pBBM::BBM:YFP somatic embryos hybridized with YFP antibody (E).
BBM
YFP
N-terminus
PKR1/
HDG11
YFP
C-terminus
protein-protein
interaction
Complete YFP
PHD zinc finger SNF2-related helicase/ATPase domain
Chromo domains DNA-binding domain
Homeobox (DNA-binding)
START domain
Table 2: Efficiency of SE induction by BnBBM and AtBBM
BBM
PKR1/
HDG11
BBM-YFP
-
control A B C D E
BBM + PKR1
BBM + HDG11