gene regulation during sexual and apomictic development in...
TRANSCRIPT
Gene regulation during sexual and apomictic development
in immature ovules of Boechera (Brassicaceae) and sorghum
John Carman
Utah State University
• Asexual seed formation (good for casual discussion)
– restricted to seed plants
– doesn’t identify mechanisms
• Meiosis replaced by apomeiosis and syngamy replaced by
parthenogenesis – better?
– yes: for gametophytic apomixis
• apomeiosis: unreduced embryo sac formation
– apospory
– diplospory
– no: for sporophytic apomixis (angiosperms) & apogamy (ferns)
• no normal apomeiosis
• no normal parthenogenesis
• In eukaryotes: life cycle renewal from a single cell without
meiosis and syngamy
Apomixis Defined
Apomixis in eukaryote kingdoms
Apomixis for Crop Improvement: Insight
from Studies of Apomixis in other Kingdoms
John Carman
Utah State University
1n
Stress
Sex and Apomixis in Haplontic Protists and Algae
No stress
Syngamy Tolerant
zygospores
Zygotic
meiosis
Apomictic
pathway
Sexual
pathway
Life cycle reset
complete
Haplontic
Meiosis
Apomeiosis
Syngamy
Parthenogenesis
Chromatin reset begins Formation of embryos and endosperm (plants)
Gamete or gametophyte (plants) formation
germ cell formation
FIG. 2. Differences in chromatin remodeling likely occur between germ cells pursuing sexual
vs. apomictic development. The extent of these differences awaits determination
2n
Sex and Apomixis in Diplontic Protists and Algae
Stress
No stress
Meiosis
Tolerant
zygospores
Syngamy
Life cycle reset
complete
Apomictic pathway
Sexual
pathway
Meiosis
Apomeiosis
Syngamy
Parthenogenesis
Chromatin reset begins Formation of embryos and endosperm (plants)
Gamete or gametophyte (plants) formation
germ cell formation
FIG. 2. Differences in chromatin remodeling likely occur between germ cells pursuing sexual
vs. apomictic development. The extent of these differences awaits determination
Diplontic
2n phase
Meiosis
Stress tolerant spores
Syngamy
-------------------------
Stress tolerant seeds
(plants)
Stress
Apomeiosis /
parthenogenesis
No stress
Eukaryote Life Cycles
FEMALE MALE
- Short
photoperiods
- Cold
temperatures
- Starvation
MALE
• Daphnia
– Most primitive metazoan
genome sequenced to date
– shares the greatest sequence
similarity with plants
• Boechera holboellii
• Calamagrostis purpurea
• Ageratina riparia
• Dichanthium aristatum
• Limonium transwallianum
• Themeda australis
• Dichanthium intermedium
• Paspalum cromyorrhizon
• Brachiaria brizantha
Angiosperms with tendencies for cyclical parthenogenesis
Cyclical Apomicts do Both
Aphids (Gallot et al., 2012 BMC Genomics) --- genes upregulated during apomeiotic phase ---
“Our results show that asexual and sexual oogenesis
in aphids share common genetic programs but
diverge by adapting specificities in their respective
gene expression profiles in germ cells and oocytes.”
1st division restitution
X
X
X
X (rare)XX
X (rare)
X
GlaucophytesRhodophytaPrasinophytesChlorophyceaeTrebouxiophyceaeUlvophyceaeChlorokybalesKlebsormidialesZygnematalesCharalesColeochaetalesMarchantiomorphaAnthocerotophytaBryophytaLycopodiopsidaPolypodiopsidaCycadsConifersGinkosGnetalesAngiosperms
Emb
ryo
ph
ytes
Sper
mat
op
sid
a
Stre
pto
ph
ytaG
ree
n p
lan
ts
1Compiled from Suomalainen et al. (1987), Bilinski et al. (1989), Asker and Jerling (1992), Mogie (1992), Bell (1992), Pressel and Duckett (2006), Nava et al. (2010)
Plantae
Form of apomeiosis1
Other
X
X
XX
X
X (rare)XX
X
Apomixis and
Plantae Phylogeny
Reversal in Panicoideae
Apomixis
Sex only
Sanchez-Ken & Clark, AJB 2010
Apospory in Sorghum (diploid, 2n = 2x = 20)
• Aposporous embryo sac (AES) formation is
common in sorghum
• Aposporously produced eggs are:
– sexually functional (triploids)
– parthenogenesis not convincingly demonstrated (in the
literature)
Apospory accelerates onset of meiosis and
sexual embryo sac formation in sorghum
From Carman et al., BMC Plant Biology 2011, 11:9
1150
K-means
clustering
Ovule stage
MMC Meiosis Early ES
Pis
til le
ngth
(µ
m)
200
300
400
500
Sib
Non-AES-forming
AES-forming
main effects (sib and stage) significant at ***; interaction significant at **
From Lacey et al., in preparation
Apospory accelerates onset of meiosis and
sexual embryo sac formation in sorghum
≈40% of ovule-expressed genes were interrogated
• The microarray (CombiMatrix)
– 12,000 ESTs (sorghum flowers)
– 11,545 (96.2%) expressed (>2 SDEV above background)
– Represents 6472 Arabidopsis genes (BLAST E-value = 5x10-2)
– 6378 genes (98.5%) expressed (2 SDEV above background)
• The experiment (3410 microexcised ovules)
– two sibs from F2 population
• aposporous (14% of ovules)
• non-aposporous
– three stages
• MMC
• Meiosis (MI&II)
• 4-nucleate ES (ES4)
– five reps (sib by stage)
– RNA extracted cDNA aRNA (Cy3 & Cy5 labeling, Ambion)
– fragmentation microarray median normalization SAM AMIGO
MMC ES4 MI&II
Transcriptome
H
M
L
Diffe
rentia
l expre
ssio
n (
324)
Non-aposporous & meiotic
Aposporous & meiotic
KEY
MMC ES4 MI&II
Transcriptome
H
M
L
Diffe
rentia
l expre
ssio
n (
324)
7 genes
down in
sexual,
no GOs
Non-aposporous & meiotic
Aposporous & meiotic
KEY
MMC ES4 MI&II
Non-aposporous & meiotic
Aposporous & meiotic
KEY
Transcriptome
H
M
L
Diffe
rentia
l expre
ssio
n (
324)
• 80 genes up in sexual ovules (seven enriched GOs) – Chromatin remodeling: 5 (HTB11, FKBP53,
AT5G59970, GEM, VIP3, DRM2)
– Organelle organization: 1
– Cellular processes: 1
• 9 genes down in sexual ovules (three enriched GOs) – Cellular processes: 1
– Organelle: 2
Aposporous MMC & MI&II (stages
1 & 2) vs. sexual MI&II (stage 2)
7 genes
down in
sexual,
no GOs
Meiosis
Apomeiosis
Syngamy
Parthenogenesis
DNA methylation reset
Formation of embryos and endosperm (plants)
Gamete or gametophyte (plants) formation
Primordial germ cell formation
MMC ES4 MI&II
Transcriptome
H
M
L
Diffe
rentia
l expre
ssio
n (
324)
7 genes
down in
sexual,
no GOs
• 11 genes up in sexual ovules (no
enriched GOs)
• 3 genes down in sexual ovules (three
enriched GOs, BIN2, FC2, PEX13)
– Protein autophosphorylation
– Protein targeting
– Intracellular protein transport
MI&II (stage 2):
aposporous vs. sexual
Non-aposporous & meiotic
Aposporous & meiotic
KEY
• 80 genes up in sexual ovules
(seven enriched GOs)
– Chromatin remodeling: 5
– Organelle organization: 1
– Cellular processes: 1
• 9 genes down in sexual ovules
(three enriched GOs)
– Cellular processes: 9
– Organelle: 2
Aposporous MMC & MI&II (stages
1 & 2) vs. sexual MI&II (stage 2)
MMC ES4 MI&II
Non-aposporous & meiotic
Aposporous & meiotic
KEY
Transcriptome
H
M
L
Diffe
rentia
l expre
ssio
n (
324)
• 2 genes up-regulated in sexual ovules (four enriched GOs) – Cell-cell junction: 3
– Symplast: 1
• 229 genes down-regulated in sexual ovules (54 enriched GOs) – Cellular processes: 22
– Organelle: 11
– Respiration: 8
– Response to various stimuli: 6
– Cell-cell transport: 3
– Transcription & translation: 3
Aposporous ES4 (stages 3)
vs. sexual ES4 (stage 3)
• 11 genes up in non-aposporous
ovules (no enriched GOs)
• 3 genes up in aposporous
ovules (three enriched GOs)
– Protein autophosphorylation
– Protein targeting
– Intracellular protein transport
MI&II: Aposporous
vs. non-aposporous
• 80 genes up in sexual ovules
(seven enriched GOs)
– Chromatin remodeling: 5
– Organelle organization: 1
– Cellular processes: 1
• 9 genes down in sexual ovules
(three enriched GOs)
– Cellular processes: 9
– Organelle: 2
Aposporous MMC & MI&II (stages
1 & 2) vs. sexual MI&II (stage 2)
Conclusions for Sorghum Expression Profiling
• Sexual ovules: 18-fold more gene-expression-dynamic
– sex: 525 genes
– apospory: 29 genes
• Response to stimuli genes
– aposporous ovules – up-regulated at all stages
– sexual ovules – up at stages 1 & 2, down at stage 3
• Differences in hormone response genes
• BIN2 (a brassinosteroid receptor – up in aposporous ovules)
• JAX1 (a JA suppressor – down in aposporous ovules)
• VIP3: down in aposporous ovules (early flowering)
– H3K4 and H3K36 methylation
– correlated with early onset of meiosis and ES formation
• GEM & DRM2: down in aposporous ovules
– H3K9 methylation
– required for meiosis
Carman JG. 2007. Regnum Vegetable 147
10 µm
MMC
20 µm
1-nucleate
embryo sacTetrad
20 µm50 µm
Mature
embryo sac
10 µm
MMC
20 µm20 µm
1-nucleate
embryo sacTetrad
20 µm50 µm
Mature
embryo sac
Sexual ovules (B. formosa)
Diplosporous
embryo sac
Apomictic ovule
(Boechera microphylla)
Aposporous
embryo sacs
A BA B
Apomictic ovules
(Boechera lignifera)
Diplosporous
embryo sacs
Expression profiling of ovules and pistils
from apomictic and sexual Boechera
John Carman
Krishna Dwivedi
Venkatesh Bhat
Balasubramanian Ganesan
Utah State University, Logan & Caisson Laboratories, Inc., North Logan
Materials and Methods
• Expression profiling: ovule experiments, 20000 ovules; pistil experiments 3000 Pistils
– Four species
• Sexual B. formosa and B. stricta
• Diplosporous & aposporous B. microphylla
• Diplosporous B. lignifera
• Four stages
– MMC (both tissues)
– Meiocyte (both tissues)
– Early embryo sac (ovules only)
– Mature embryo sac (ovules only)
• RNA and array procedures
– Ovule collected in RNAlater (Ambion)
– RNA extraction
• TRIzol reagent (Invitrogen)
• Cleanup (Qiagen)
• RNA yield (NanoDrop)
– Two-cycle cDNA synthesis followed by cRNA amplification
– ATH1 Gene Chip® arrays and analyses (Affymetrix)
• Data analyses
– Normalized across samples (RMA preprocessing algorithm)
– Comparisons made using SAM software
– Enriched gene ontology (GO) categories identified using GOEAST
1
2
3
4
Stage Pistil
length
(mm)
Germline stage
1 1.3 MMC/meiocyte
2 1.8 Meiocyte – ES1
3 2.5 ES2-early ES8
4 5.5 Mature ES8
Sexual Apomictic
Up-r
egula
ted g
enes
200
400
600
800
1000
1200
1400
MMC Formation through ES Development
(Sexual B. stricta vs. apomictic B. lignifera)
B. stricta: 176 GO
• Signaling - 2
• Response to stimuli – 25
• REDOX signaling – 12
• Other – 13
• Protein synthesis/catabolism – 47
• Meiosis – 8
• Development – 5
• Other functions - 89
B. lignifera: 203 GO
• Signaling - 3
• sRNA mediated gene silencing - 5
• Response to stimuli – 11 (light – 7)
• Photosynthesis – 26
• Development – 9
• Other cellular activities – 140
1346
1464
Pistils
Genes: 2810 Enriched GO: 379
Intracellular Signal Transduction – Up in Sexual
probeset_ID Target Description Gene Symbol
246422_atADP-ribosylation factor -like protein ADP-ribosylation factor, Dugesia japonica, EMBL:DJAB1051;supported by
full-length cDNA: Ceres:26844.ATARFB1B
265337_at putative ADP-ribosylation factor ;supported by full-length cDNA: Ceres:5695. TTN5
258656_atputative Ras-like GTP-binding protein contains Pfam profile: PF00071 Ras family;supported by full-length cDNA:
Ceres:10506.ATRABE1E
256129_atcalcium-binding protein, putative similar to calcium-binding protein GI:6901652 from [Olea
europaea];supported by full-length cDNA: Ceres:19462.---
253757_at rac GTP binding protein Arac7 ; supported by cDNA: gi_3702961_gb_AF079484.1_AF079484 ROP9
262729_atrac-like GTP binding protein (ARAC5) identical to rac-like GTP-binding protein (ARAC5) SP:Q38937 [Arabidopsis
thaliana (Mouse-ear cress)]; supported by cDNA: gi_1293667_gb_U52350.1_ATU52350ARAC5
254926_at ACC synthase (AtACS-6) ; supported by cDNA: gi_16226285_gb_AF428292.1_AF428292 ACS6
246290_at calmodulin-3 ;supported by full-length cDNA: Ceres:16715. CAM3
257754_atGTP binding protein, putative similar to RAS-RELATED PROTEIN RAB7 GB:P31022 from [Pisum sativum], Plant
Mol. Biol. 21 (6), 1195-1199 (1993);supported by full-length cDNA: Ceres:101693.ATRAB7B
251960_at GTPase AtRAB8 ;supported by full-length cDNA: Ceres:27384. HEMB1
251158_atrac-GTP binding protein -like RACC small GTP binding protein, Zea mays, EMBL:AF126054; supported by cDNA:
gi_16648802_gb_AY058178.1_MIRO2
245251_at calcineurin B-like protein 1 ; supported by cDNA: gi_3309081_gb_AF076251.1_AF076251 CBL1
256397_atputative dual-specificity protein phosphatase similar to dual-specificity protein phosphatase GB:CAA77232
[Arabidopsis thaliana];supported by full-length cDNA: Ceres:17858.MKP2
245407_at GTP-binding RAB2A like protein RABB1C
263529_at ADP-ribosylation factor 3 ARF3
259395_atGTP-binding protein(RAB11D), putative similar to RAB11D GI:1370148 from [Lotus japonicus]; supported by
cDNA: gi_12083263_gb_AF332428.1_AF332428ARA-2
261069_at small G protein, putative similar to GB:Z49190 from [Beta vulgaris] ATRABA2B
GO Related to Oxidative Stress
S up TermNumber in
your list
Number on
Gene Chip® p-value
S1 response to stress 135 2116 0.000961
S1 response to oxidative stress 29 290 0.00638
S1 respiratory chain 21 90 1.27E-07
S1S2 respiratory chain 11 90 0.00356
S1 respiratory chain complex I 11 50 0.00187
S1S2 respiratory chain complex I 8 50 0.00706
S1 mitochondrial respiratory chain 11 61 0.0112
S1 ubiquinol-cytochrome-c reductase activity 4 9 0.0586
S1 NADH dehydrogenase (ubiquinone) activity 6 18 0.0146
S1 NADH dehydrogenase (quinone) activity 6 18 0.0146
S1oxidoreductase activity, acting on NADH or NADPH, quinone or similar
compound as acceptor8 29 0.00511
S1S2oxidoreductase activity, acting on NADH or NADPH, quinone or similar
compound as acceptor5 29 0.0993
S1oxidoreductase activity, acting on diphenols and related substances as
donors, cytochrome as acceptor4 9 0.0586
S1 NADH dehydrogenase complex 11 50 0.00187
S1S2 NADH dehydrogenase complex 8 50 0.00706
S1 NADH dehydrogenase activity 7 25 0.0133
probeset_ID Target Description Gene Symbol
266294_at putative small heat shock protein ;supported by full-length cDNA: Ceres:25828. ---
254386_at peroxidase prxr1 ;supported by full-length cDNA: Ceres:20758. PRXR1
265675_at 70kD heat shock protein ;supported by full-length cDNA: Ceres:98979. HSP70T-2
252592_at mitogen-activated protein kinase 3 ; supported by cDNA: gi_14423447_gb_AF386961.1_AF386961 NAXT1
260248_at heat shock protein 101 (HSP101) identical to heat shock protein 101 GI:6715468 GB:AAF26423 from [Arabidopsis thaliana] ATHSP101
263150_at heat-shock protein, putative similar to heat-shock protein GI:472939 from [Helianthus annuus];supported by full-length cDNA: Ceres:97415. ---
256245_at heat shock protein 70 identical to heat shock protein 70 GB:CAA05547 GI:3962377 [Arabidopsis thaliana]; supported by cDNA: gi_15809831_gb_AY054183.1_ HSP70
262911_s_at heat shock protein, putative similar to heat shock protein GI:19617 from [Medicago sativa];supported by full-length cDNA: Ceres:32795. ---
264809_at superoxidase dismutase identical to GB:P24704;supported by full-length cDNA: Ceres:33493. CSD1
249575_at low-molecular-weight heat shock protein - like cytosolic class I small heat-shock protein HSP17.5, Castanea sativa, EMBL:CSA9880 ---
261518_at peroxidase ATP4a identical to GB:CAA67309 GI:1429213 from [Arabidopsis thaliana]; supported by full-length cDNA: Ceres: 39968. ---
255891_at hypothetical protein predicted by genscan+ EGY3
258851_at glutathione S-transferase identical to glutathione S-transferase GB:AAB09584 from [Arabidopsis thaliana]; supported by cDNA: gi_1575751_gb_U70672.1_ATU70672 ATGSTF11
265735_at putative aldolase ;supported by full-length cDNA: Ceres:22418. ---
246870_at ferrochelatase-I FC1
249344_at prohibitin (gb AAC49691.1) ;supported by full-length cDNA: Ceres:37298. ATPHB3
260746_at glutathione transferase, putative similar to glutathione transferase GI:2853219 from [Carica papaya]; supported by full-length cDNA: Ceres: 30759. ATGSTU19
266841_at putative heat shock transcription factor ATHSFA2
258939_at unknown protein predicted by genefinder, multiple est matches;supported by full-length cDNA: Ceres:7073. ---
AFFX-Athal-
GAPDH_5_s_at
Arabidopsis thaliana /REF=M64116 /DEF=glyceraldehyde 3-phosphate dehydrogenase C subunit (GapC) gene, complete cds /LEN=1295 (_5, _M, _3 represent transcript regions
5 prime, Middle, and 3 prime respectively)GAPC1
250928_at putative protein EIN2
267357_at putative nematode-resistance protein ;supported by full-length cDNA: Ceres:35056. HSPRO2
254926_at ACC synthase (AtACS-6) ; supported by cDNA: gi_16226285_gb_AF428292.1_AF428292 ACS6
AFFX-Athal-
GAPDH_M_s_at
Arabidopsis thaliana /REF=M64116 /DEF=glyceraldehyde 3-phosphate dehydrogenase C subunit (GapC) gene, complete cds /LEN=1295 (_5, _M, _3 represent transcript regions
5 prime, Middle, and 3 prime respectively)GAPC1
267101_at putative peroxidase ---
258606_at unknown protein ;supported by full-length cDNA: Ceres:38495. ---
259361_atglyceraldehyde-3-phosphate dehydrogenase, putative similar to glyceraldehyde-3-phosphate dehydrogenase GI:21143 from (Sinapis alba); supported by cDNA:
gi_15146235_gb_AY049259.1_GAPC2
256397_at putative dual-specificity protein phosphatase similar to dual-specificity protein phosphatase GB:CAA77232 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:17858. MKP2
251984_at phenylalanine ammonia-lyase ;supported by full-length cDNA: Ceres:110886. PAL2
247691_at heat shock protein 18 ;supported by full-length cDNA: Ceres:97197. HSP18.2
266001_at hypothetical protein predicted by genefinder; supported by cDNA: gi_13877832_gb_AF370179.1_AF370179 HHP3
263426_at putative glutathione peroxidase ;supported by full-length cDNA: Ceres:25550. ATGPX2
Response to Oxidative Stress – Up in Sexual
Meiosis Genes – Up in Sexual (Down in Apomict)
probeset_ID Target DescriptionGene
Symbol
248658_at chromosome condensation protein ATSMC3
252736_at kinesin -like protein kinesin-like protein ZCF125, Arabidopsis thaliana, EMBL:AB028468 TES
256832_at
meiotic recombination protein (AtDMC1) identical to AtDMC1 GB:AAC49617 [Arabidopsis
thaliana] (Plant J. 11 (1), 1-14 (1997)) contains non-consensus AT donor splice site at exon
14
DMC1
254400_at kinesin-related protein katA ; supported by cDNA: gi_303501_dbj_D11371.1_ATHKATA ATK1
247301_at DNA topoisomerase III ---
265678_atputative RAD50 DNA repair protein ; supported by cDNA:
gi_7110147_gb_AF168748.1_AF168748RAD50
247482_atchromosomal protein - like chromosomal protein XCAP-E, Xenopus laevis, PIR:B55094;
supported by cDNA: gi_12382275_gb_AF306547.1_AF306547SMC2
Sexual Apomictic
Up
-re
gu
late
d g
en
es
500
1000
1500
2000
2500
3000
3500
B. formosa: 577 GO
• Stress & other stimuli – 109
• REDOX signaling – 42
• Jasmonic acid signaling – 4
• Other stress/response GO - 63
• Nucleotides nucleosides – 30
• Photosynthesis – 26
• Development – 29
• Male - 6
• Other cellular activities – 386
B. lignifera: 236 GO
• sRNA mediated gene silencing – 50
• Chromatin remodeling
• Post-transcriptional
• Cell cycle regulation – 25
• Floral development - 28
• Other cellular activities – 134
MMC Formation through ES Development
(sexual B. formosa vs. apomictic B. lignifera)
2351
3434
Ovules
Genes: 5785 Enriched GO: 813
probeset_ID Target Description Gene Symbol
266294_at putative small heat shock protein ;supported by full-length cDNA: Ceres:25828. ---
254386_at peroxidase prxr1 ;supported by full-length cDNA: Ceres:20758. PRXR1
265675_at 70kD heat shock protein ;supported by full-length cDNA: Ceres:98979. HSP70T-2
252592_at mitogen-activated protein kinase 3 ; supported by cDNA: gi_14423447_gb_AF386961.1_AF386961 NAXT1
260248_at heat shock protein 101 (HSP101) identical to heat shock protein 101 GI:6715468 GB:AAF26423 from [Arabidopsis thaliana] ATHSP101
263150_at heat-shock protein, putative similar to heat-shock protein GI:472939 from [Helianthus annuus];supported by full-length cDNA: Ceres:97415. ---
256245_at heat shock protein 70 identical to heat shock protein 70 GB:CAA05547 GI:3962377 [Arabidopsis thaliana]; supported by cDNA: gi_15809831_gb_AY054183.1_ HSP70
262911_s_at heat shock protein, putative similar to heat shock protein GI:19617 from [Medicago sativa];supported by full-length cDNA: Ceres:32795. ---
264809_at superoxidase dismutase identical to GB:P24704;supported by full-length cDNA: Ceres:33493. CSD1
249575_at low-molecular-weight heat shock protein - like cytosolic class I small heat-shock protein HSP17.5, Castanea sativa, EMBL:CSA9880 ---
261518_at peroxidase ATP4a identical to GB:CAA67309 GI:1429213 from [Arabidopsis thaliana]; supported by full-length cDNA: Ceres: 39968. ---
255891_at hypothetical protein predicted by genscan+ EGY3
258851_at glutathione S-transferase identical to glutathione S-transferase GB:AAB09584 from [Arabidopsis thaliana]; supported by cDNA: gi_1575751_gb_U70672.1_ATU70672 ATGSTF11
265735_at putative aldolase ;supported by full-length cDNA: Ceres:22418. ---
246870_at ferrochelatase-I FC1
249344_at prohibitin (gb AAC49691.1) ;supported by full-length cDNA: Ceres:37298. ATPHB3
260746_at glutathione transferase, putative similar to glutathione transferase GI:2853219 from [Carica papaya]; supported by full-length cDNA: Ceres: 30759. ATGSTU19
266841_at putative heat shock transcription factor ATHSFA2
258939_at unknown protein predicted by genefinder, multiple est matches;supported by full-length cDNA: Ceres:7073. ---
AFFX-Athal-
GAPDH_5_s_at
Arabidopsis thaliana /REF=M64116 /DEF=glyceraldehyde 3-phosphate dehydrogenase C subunit (GapC) gene, complete cds /LEN=1295 (_5, _M, _3 represent transcript regions
5 prime, Middle, and 3 prime respectively)GAPC1
250928_at putative protein EIN2
267357_at putative nematode-resistance protein ;supported by full-length cDNA: Ceres:35056. HSPRO2
254926_at ACC synthase (AtACS-6) ; supported by cDNA: gi_16226285_gb_AF428292.1_AF428292 ACS6
AFFX-Athal-
GAPDH_M_s_at
Arabidopsis thaliana /REF=M64116 /DEF=glyceraldehyde 3-phosphate dehydrogenase C subunit (GapC) gene, complete cds /LEN=1295 (_5, _M, _3 represent transcript regions
5 prime, Middle, and 3 prime respectively)GAPC1
267101_at putative peroxidase ---
258606_at unknown protein ;supported by full-length cDNA: Ceres:38495. ---
259361_atglyceraldehyde-3-phosphate dehydrogenase, putative similar to glyceraldehyde-3-phosphate dehydrogenase GI:21143 from (Sinapis alba); supported by cDNA:
gi_15146235_gb_AY049259.1_GAPC2
256397_at putative dual-specificity protein phosphatase similar to dual-specificity protein phosphatase GB:CAA77232 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:17858. MKP2
251984_at phenylalanine ammonia-lyase ;supported by full-length cDNA: Ceres:110886. PAL2
247691_at heat shock protein 18 ;supported by full-length cDNA: Ceres:97197. HSP18.2
266001_at hypothetical protein predicted by genefinder; supported by cDNA: gi_13877832_gb_AF370179.1_AF370179 HHP3
263426_at putative glutathione peroxidase ;supported by full-length cDNA: Ceres:25550. ATGPX2
Response to Oxidative Stress
probeset_ID Target DescriptionGene
Symbol
266314_at Argonaute (AGO1)-like protein ; supported by cDNA: gi_14334815_gb_AY035081.1_ AGO4
267641_at Argonaute (AGO1)-like protein AGO6
256293_at pinhead-like protein similar to pinhead [Arabidopsis thaliana] GI:5107374 AGO7
258848_at Expressed protein ; supported by full-length cDNA: Ceres: 9573. DCL2
252716_at putative protein CAF protein, Arabidopsis thaliana, EMBL:AF187317 DCL3
248438_at putative protein contains similarity to fertilization-independent seed 2 protein EMF2
249816_at cleavage and polyadenylation specificity factor ; supported by cDNA: gi_14334617_gb_AY034982.1_ CPSF100
261903_atchromomethylase identical to GB:AAC02660 GI:2865416 from [Arabidopsis thaliana]; supported by cDNA:
gi_2766712_gb_AF039364.1_AF039364CMT1
250139_at putative protein de novo DNA methyltransferase 3, Danio rerio, EMBL:AF135438 DRM2
250140_at putative protein DRM2
249036_atnuclear cap-binding protein; CBP20 (gb|AAD29697.1) non-consensus AT donor splice site at exon 4, AC acceptor splice site at exon 5;
supported by cDNA: gi_4768967_gb_AF140219.1_AF140219CBP20
252637_at WD repeat domain protein nuclear protein HIRA, mouse, PIR:S68141 HIRA
247056_at SWI2/SNF2-like protein (gb|AAD28303.1) CHR1
254933_at putative RNA-directed RNA polymerase RNA-directed RNA polymerase - Nicotiana tabacum, PID:e1363996 RDR2
252261_at RNA-directed RNA polymerase RNA-directed RNA polymerase RDR6
258148_s_atDNA-directed RNA polymerase II second largest chain, putative similar to DNA-directed RNA polymerase II second largest chain
GB:S30229 [Arabidopsis thaliana]NRPD2B
249457_s_at zinc finger -like protein transcription factor ICBP90, Homo sapiens, EMBL:AF129507 VIM4
263727_at putative cap-binding protein ; supported by cDNA: gi_15192737_gb_AF272891.1_AF272891 ABH1
267350_at putative DNA-directed RNA polymerase II subunit NRPD1B
267351_at unknown protein NRPD1B
264748_at hypothetical protein predicted by genemark.hmm EMB25
248030_at transcription regulator Sir2-like protein ; supported by cDNA: gi_12006419_gb_AF283757.1_AF283757 SRT1
247972_at histone acetyltransferase HAT B HAG2
252254_at hypothetical protein ; supported by cDNA: gi_15146293_gb_AY049288.1_ DMS3
260625_atstorage protein, putative similar to GB:CAA53781 from [Dioscorea cayenensis] (Plant Mol. Biol. 28 (3), 369-380 (1995)); supported by
cDNA: gi_8132767_gb_AF213627.1_AF213627MOM
266539_at similar to mammalian MHC III region protein G9a ; supported by cDNA: gi_13517750_gb_AF344448.1_AF344448 SUVH5
251233_at putative protein hypothetical protein At2g28380 - Arabidopsis thaliana, EMBL:AC006283 DRB4
260417_at putative chromomethylase similar to chromomethylase GB:AAB95486 [Arabidopsis arenosa] CMT3
Regulation of Gene Expression, Epigenetic
Sexual Apomictic
Up
-re
gu
late
d g
en
es
500
1000
1500
2000
2500
3000
3500
4000
B. formosa: 497 GO
• Stress & other stimuli – 76
• REDOX signaling – 26
• Jasmonic acid signaling – 6
• Other stress/response GOs - 44
• Nucleotides nucleosides – 37
• Photosynthesis – 23
• Development – 20 (maleness – 2)
• Other cellular activities – 345
B. microphylla: 305 GO
• Signaling (shade avoidance) – 1
• sRNA mediated gene silencing – 48
• Chromatin remodeling
• Posttranscriptional
• Regulation of cell cycle – 22
• Development – 48 (embryo - 8)
• Other cellular activities – 187
MMC Formation through ES Development
(sexual B. formosa vs. apomictic B. microphylla)
3811
2622
Ovules
Genes: 6433 Enriched GO: 802
Conclusions
• Apomixis: life cycle renewal from single cells without meiosis and
syngamy
• Sexual and apomictic development in aphids, sorghum and Boechera
involves differential expression of:
– inter and intra-cellular signal transduction genes
– chromatin remodeling genes
– post-transcriptional gene regulation genes
• The existence of an ancient sex/apomixis switch is inferred
• The switch is more or less conserved in all eukaryotes
– Highly conserved in cyclical apomicts
– Moderately conserved in facultative apomicts
– Poorly conserved in obligate sexual or obligate apomictic
organisms
• Domestication of apomixis for crop improvement
– elucidate the mechanisms of apomixis
– Elucidate the mechanisms of the sex apomixis switch
Project conception: John Carman, Jeff Lacey,
Krishna Dwivedi, Venkatesh Bhat, Jayasree
Pattanayak, Michelle Jamison
Array design and construction: Jeff Lacey and
Tom Ulrich
Embryological analyses and dissections:
Michelle Jamison, Becky Kowallis, Suzette
Hatch, Paul Daybell
Molecular tests and data analysis: Jeff Lacey,
Krishna Dwivedi, Venkatesh Bhat, Jonathan
Cardwell, John Carman, Estella Elliot
Manuscript preparation: Jeff Lacey, John
Carman, Jonathan Cardwell, Krishna Dwivedi,
Venkatesh Bhat
Funding
• NIST, ATP
• Utah Agri Exp Station
• Caisson Laboratories, Inc.
Acknowledgments