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The life cycle of Arabidopsis thaliana
Nuclear Transcriptome Analyses of Specific
Cell Types in the Early Plant Embryo
Daniel Slane
MPI for Developmental Biology
Tuebingen, Germany
12.01.2016
Available applications for tissue-specific omics
FACS as a successful method applied in Arabidopsis
Laser capture microdissection
Translating ribosome affinity purification
Fluorescence-activated cell sorting
Isolation of nuclei tagged in specific cell types
Yadav et al., 2014
Siobhan et al., 2007
Borges et al., 2012
Palovaara et al., 2013
FANS (Fluorescence-activated nuclear sorting)
Arabidopsis early embryogenesis – underlying mechanisms
Lau et al. 2012
Zhang et al., 2008
Lau et al., 2012
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Tissue-specific marker lines
pAT5G42200:n3xGFP
Suspensor
pDRN:n2xGFP:DRN 3’UTR
Proembryo
FANS with established marker lines
no marker
suspensor marker
proembryo marker
whole embryo marker
pAT3G10010:nGFP
Whole Embryo
→ droplets caught directly in
RNA extraction buffer
Validity of nuclear expression data
Protocol overview and microarray hybridization
Cell type-specific marker line
Sorting of fluorescent nuclei
RNA extraction, RNA amplification and RNA labeling
r = 0.97
Means normalized values cEMB
Fixation and extraction of nuclei
Microarray hybridization in triplicates
Means normalized values nEMB
Slane et al., 2015
Microarray analysis
Gene Ontology (GO) categories
Proembryo enriched
Suspensor enriched
• 34% (whole embryo), 32% (proembryo) and 25% (suspensor) of
transcripts are 3x present in all samples (MAS5 algorithm)
• 307 transcripts are more than 2-fold enriched in the pro-embryo
samples as compared to the suspensor samples (gcRMA algorithm)
• 180 transcripts are more than 2-fold enriched in the suspensor samples
as compared to the pro-embryo samples (gcRMA algorithm)
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Differentially expressed genes previously described
Confirmation of expression data in vivo
• Genes involved in auxin biosynthesis (TAA1, YUC4), transport (PIN1)
and signal transduction (SHI, MYB77)
• Genes important for embryo development in general (WOX2, HAN, OBP1, FUS3)
HANABA TARANU (HAN)
A
pAT3G62480::n3GFP
B
pAT3G52780::n3GFP
C
pAT2G32100::n3GFP
OBF BINDING PROTEIN 1 (OBP1)
D
AT1G04645
antisense
Nawy et al., 2012
E
pAT5G05940::n3GFP
F
pAT3G17290::n3RFP
sense
G
AT1G28300
antisense
sense
Skirycz et al., 2008
Overview of in vivo validation differentially expressed genes
Possible redundancies during early embryogenesis
Proembryo
Fold change
3,92
3,14
Locus Identifier
AT5G61030
AT3G23830
Annotation
GR-RBP3 (glycine-rich RNA-binding protein 3); RNA binding
GR-RBP4/GRP4 (GLYCINE-RICH RNA-BINDING PROTEIN 4)
6,50
2,22
AT5G05940
AT3G55660
ATROPGEF5/ROPGEF5 (KINASE PARTNER PROTEIN-LIKE)
ATROPGEF6/ROPGEF6 (KINASE PARTNER PROTEIN-LIKE)
3,41
2,31
AT5G22650
AT3G44750
HD2B (HISTONE DEACETYLASE 2B)
HD2A (HISTONE DEACETYLASE 2A)
X
2,87
2,83
AT4G35570
AT2G17560
HMGB5 (HIGH MOBILITY GROUP B 5); transcription factor
HMGB4 (HIGH MOBILITY GROUP B 4); transcription factor
X
2,24
2,43
AT3G50410
AT5G66940
OBP1 (OBF BINDING PROTEIN 1)
Dof-type zinc finger domain-containing protein
Fold change
7,92
2,56
Locus Identifier
AT3G04070
AT1G69490
Annotation
ANAC047 (Arabidopsis NAC domain containing protein 47)
NAP (NAC-LIKE, ACTIVATED BY AP3/PI); transcription factor
2,84
2,73
AT2G32100
AT1G79960
ATOFP16/OFP16 (Arabidopsis thaliana ovate family protein 16)
ATOFP14/OFP14 (Arabidopsis thaliana ovate family protein 14)
3,55
2,83
AT2G02990
AT1G26820
RNS1 (RIBONUCLEASE 1); endoribonuclease
RNS3 (RIBONUCLEASE 3); endoribonuclease
4,39
2,13
AT1G54160
AT5G47670
CCAAT-binding transcription factor (CBF-B/NF-YA) family protein
CCAAT-box binding transcription factor family protein
Proembryo enriched transcripts tested
Locus
Promoter expression analysis in vivo
AT5G26270
AT1G77580
AT5G05940
AT3G17290
AT2G35605
AT5G61030
AT1G31400
AT1G64220
AT1G28300
AT5G22650
AT5G43510
AT3G44750
AT3G55660
EMB, stronger PE
globular stage PE
EMB, at late globular/early heart stage stronger PE
EMB, at 8/16-cell stage stronger PE
EMB, stronger PE
EMB, stronger PE
no expression
globular stage PE
not available
inconsistant expression
PE early heart stage
inconsistant expression
late globular stage hypophysis and lower tier
In situ
X
X
Suspensor enriched transcripts tested
Suspensor
Locus
Promoter expression analysis in vivo
In situ
AT2G46690
AT3G62480
AT1G48470
AT1G04645
AT1G54160
AT3G52780
AT5G46230
AT5G07440
AT1G74190
AT2G32100
no expression
SUS
no expression
not available
EMB, stronger SUS
SUS
EMB, stronger SUS
EMB, stronger SUS
SUS
EMB, stronger SUS
X
X
X
Reliability of nuclear expression data
nEMB = nuclei whole embryo
PCA (Principal component analysis
cEMB = cells whole embryo
nPE = nuclei proembryo
nSUS = nuclei suspensor
cgPE = cells globular proembryo
cgSUS = cells globular suspensor
Principal component 2
MAS5 3x Present
Differences between embryonic expression data
Principal component 1
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Embryonic protoderm – the epidermis precursor
Advantage of FANS over LCM
RPK1
ACR4
Endosperm-specific genes
Gene title
MAS5 call nSUS
ARF12 (AUXIN RESPONSE FACTOR 12); transcription factor
A
P
ARF17 (AUXIN RESPONSE FACTOR 17); transcription factor
A
P
ARF21 (AUXIN RESPONSE FACTOR 21); transcription factor
A
P
ARF23 (AUXIN RESPONSE FACTOR 23); transcription factor
A
P
MEA (MEDEA); sequence-specific DNA binding / transcription factor
A
P
FIS2 (FERTILIZATION INDEPENDENT SEED 2); transcription factor
A
P
FWA; DNA binding / protein binding / transcription factor
P
P
MAS5 call cGSUS
AGL62 (Agamous-like 62); DNA binding / transcription factor
P
P
CKX2 (CYTOKININ OXIDASE 2); amine oxidase/ cytokinin dehydrogenase
A
P
MYB118; DNA binding / transcription activator/ transcription factor
A
P
MYB118; DNA binding / transcription activator/ transcription factor
A
P
Nuclear RNA for RNA sequencing
RNA (RIN 6.6)
WOX
PDF2
TOAD2
ALE2
ATML1
receptor-like kinases
?
8-cell stage
16-cell stage
globular stage
Summary
Pre-amplification with SmartSeq2
• Inaccessible tissue-types, few or even single cells
• Detection of tissue-specific genes
• Less prone to contamination
RNAseq library
• Not restricted to plant organisms
• DNA and histone modifications
Acknowledgements
Gerd Jürgens
Jixiang Kong
Ive De Smet
Ulrike Mayer
Martina Kolb
Martin Bayer
Agnes Henschen
ZMBP facility for Flow Cytometry
Kenneth Berendzen
Joachim Kilian
Klaus Harter
Markus Schmid
MPI Department V
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