Download Session 6: Embryogenesis

Embryogenesis session P-­‐EMB1 A framework for cell-autonomous specification of multiple tissue identities
in the early Arabidopsis embryo
Colette A. ten Hove1*, Barbara K. Möller1,#*, Doaquan Xiang2, Nerys A.
Williams1,§, Lorena González López1, Margot E. Smit1, Raju Datla2 and Dolf
Weijers1,*
1
Laboratory of Biochemistry, Wageningen University, Wageningen, the Netherlands
2
Plant Biotechnology Institute, National Research Council, Saskatoon, Saskatchewan,
Canada
#
Current address: Department of Plant Systems Biology, VIB, Ghent University, Ghent,
Belgium, Department of Plant Biotechnology and Bioinformatics, Gent University, Ghent,
Belgium
§
Current address: Hubrecht Institute, Utrecht, the Netherlands
*These authors contributed equally
Plant organs are typically organized into three main tissue layers. The middle ground
tissue layer comprises the majority of the plant body and serves a wide range of
functions, including photosynthesis, selective nutrient uptake and storage, and gravity
sensing. Ground tissue patterning and maintenance in Arabidopsis are controlled by a
well-established gene network revolving around the key regulator SHORT-ROOT (SHR).
In contrast, it is completely unknown how tissue identity is first specified from totipotent
precursor cells in the embryo. The plant signaling molecule auxin, acting through AUXIN
RESPONSE FACTOR (ARF) transcription factors, is critical for embryo patterning. The
auxin effector ARF5/MONOPTEROS (MP) acts both cell-autonomously and non-cell
autonomously to control embryonic vascular tissue formation and root initiation,
respectively. Here, we identified embryonic MP target genes through transcriptome
profiling upon local inhibition and large-scale validation. We show that, in addition to its
well-established roles, MP is also required to transcriptionally initiate the first embryonic
ground tissue cells. Strikingly, while the SHR network depends on MP, this novel MP
function is, at least in part, SHR-independent. Moreover, mp mutant embryos, but not
SHR-network mutants, show division defects in the first ground tissue precursor cells.
Local ARF inhibition in either vascular or ground tissue precursors showed that auxin
promotes specification of both tissues in a cell-autonomous manner. Our study therefore
identifies the first regulator of ground tissue specification in the embryonic root, and
provides a framework for tissue patterning through multiple, local auxin responses.
P-­‐EMB2 Embryogenesis session Arabidopsis NHP247 is required for cytokinesis in early embryogenesis
through its actin bundling activity
Ya-Wen Hsu1, 2, Huei-Jing Wang1, Guang-Yuh Jauh3, 2, 3
1
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 11529,
Taiwan.
2
Institute of Plant Biology, National Taiwan University, Taipei 116, Taiwan.
3
Biotechnology Center, National Chung-Hsing University, Taichung, 402, Taiwan.
Cytokinesis is the final step to separate the forming daughter cells and multiple biological
processes including cytoskeletal dynamic and vesicle trafficking are involved. However,
the precise molecular mechanism underneath this important process is still under
exploration. Previous in silico screening of Arabidopsis T-DNA-insertional mutants with
defects in diverse processes during reproduction, several novel nhp (no homozygote
progeny) mutants were found and one of them, nhp247, showed defective early
embryogenesis. NHP247 encodes an unknown function protein and is universally found
in all bilaterians and plants but not in unicellular yeast. Progeny of self-pollination
nhp247 heterozygote showed a quarter of embryos with developmental arresting at
globular stage that could be fully rescued by its GFP-tagged genomic fragment. In
prophase, NHP247 is ubiquitously present in cytosol whereas cells exhibited a
perinuclear microtubule array and condensed chromosomes, then NHP247-GFP started
to co-localize with phragmoplast until end of cytokinesis. To identify the promising
NHP247 interacting-proteins, affinity purification and LC-MSMS analysis of total proteins
from
complemented
lines
were
applied.
Several
actin
isoforms
were
co-
immunoprecipitated with NHP247 and their interactions were further validated by
immunoblotting. Interestingly, the co-sedimentation analyses showed that under certain
ranges of [Ca2+] and pH NHP247 stimulated F-actin bundling as evidenced by both
phalloidin-staining and in vitro immunofluorescent assays. PIN1, the major auxin
transport protein in globular embryo, were missed-accumulated at upper tier and the
provascular cells in nhp247. Our results uncover that NHP247 F-actin is an acting
bundling protein and plays vital role in properly targeting of plasmamembrane/proteins to
developing cell plate during cytokinesis.
Embryogenesis session P-­‐EMB3 Regulation of miR156 in embryos of Arabidopsis thaliana by the CDK8 module of
Mediator
Danya Castro-Echeverría, Alma Armenta-Medina, Rocío García-Flores, C. Stewart
Gillmor
Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de
Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAVIPN), Irapuato, Guanajuato
miR156 is one of the most highly expressed and deeply conserved microRNAs in plants.
This miRNA has been demonstrated to regulate the expression of seed storage protein
genes in embryogenesis, as well as post-embryonic traits such as the timing of leaf
initiation and the onset of phase-specific leaf traits. In vegetative and reproductive
tissues, miR156 levels are regulated by GRAND CENTRAL CITY (GCT)/MED13 and
CENTRAL CITY (CCT)/MED12, two subunits of the CDK8 module of Mediator which are
also known to control the timing of pattern formation in embryogenesis. We are currently
exploring the hypothesis that GCT and CCT regulation of pattern formation occurs
through miR156. We have found that miR156 levels are higher in gct and cct mutants
compared to wt embryos, suggesting that miR156 is negatively regulated by GCT and
CCT in embryos as well as in post-embryonic development.Transgenic overexpression
of miR156 produces embryo phenotypes similar to the gct and cct mutants. We are
currently carrying out experiments to determine if the misregulation of miR156 in gct and
cct embryos is the cause of the morphological phenotype of these mutants.
Embryogenesis session P-­‐EMB4 A multi-targeted protein DEE plays an essential role in embryo-endosperm
communication
Sheng Zhong, Jia Wei, Lulu Liu, Li-jia Qu
Peking University, China
Co-ordination between the embryo and the endosperm is required for seed
development, which involves intensive communication between the two tissues.
Here we report that an essential gene DEE (defective embryo and endosperm) plays an
active role in embryo/endosperm communication. The dee-/- mutant was embryo lethal,
in which both embryo and endosperm development were arrested at very early stage.
DEE protein targeted to multiple compartments, i.e., localized in cytoplasm, mitochondria
and chloroplast. Only cytoplasm version could completely rescue the embryo lethal
phenotype, suggesting that DEE localized in cytoplasm was sufficient for embryo and
endosperm development. Embryo-specific expression of DEE could largely rescue the
embryo defect phenotype of the dee-/- mutants, but not the endosperm defects.
Interestingly, however, endosperm-specific expression of DEE could rescue the
defective phenotypes both in embryo and endosperm, suggesting that DEE-mediated
production of essential substances was transported from endosperm to embryo. This
result demonstrates that DEE plays an essential role in communication between
endosperm and embryo.