From genes and molecular mechanisms to ecological, evolu4onary and biotechnological implica4ons Victor Busov Michigan Technological University vbusov@mtu.edu Genes are founda4onal …... Ecosystems Managed and/Unmanaged Ecosystem and evolu@onary processes Gene@c improvement/Biotechnology Organisms Heredity in a broader sense -­‐ communi@es Tissues/Organs Heredity in a narrow sense Metabolites Cells Proteins RNA Genes Eco-­‐devo Evo-­‐devo Biotechnology Molecular mechanisms BUT…..are we there yet? •
•
Ecosystems Managed and Unmanaged •
Organisms •
Tissues/Organs •
Cells Metabolites •
Proteins •
RNA Genes Expensive and long-­‐term Administra@vely burdensome because involve GMOs Social context – nega@ve percep@on driven by distrust, fear and ignorance LiDle known about the genes that are linked to ‘ecosystem and evolu4onary traits’ Transla4onal approaches are s4ll difficult Most of where my research focuses at the moment Baby steps to ‘break the ceiling’ and scale my research to ecosystem scale via: •  Iden@fying genes underlying adap@ve traits •  Small scale field trials •  Licensing patents to companies that have resources to do meaningful field trials Tools and approaches Proteomics OMICS Metabolomics Transcriptomics Other ‘omics’ Genomics Modeling and inferences Valida@on System knowledge Advanced breeding and management prac@ces Gene@cs Why Trees? Sol$s PS, Sol$s DE, Chase MW,1999. Nature •  Essential for
understanding plants
in general – ancestral
to all seed plants
•  First angiosperm
Amborella was shrub
•  Trees dominate
terrestrial ecosystems
and thus economically
and environmentally
relevant
Extensive hybrid poplar planta4ons in eastern Oregon Traits of interest Crown architecture Maturation
Cambium activity
Dormancy
Wood Formation
Stem
Elongation
Root development
Tools and approaches Proteomics Metabolomics Transcriptomics Other ‘omics’ Genomics Modeling and inferences Valida@on System knowledge Advanced breeding and management prac@ces Gene@cs Forward and Reverse Gene4cs Phenotype FORWARD GENETICS ‘BIG TREE’ ??? Phenotype Genotype ??? Gene (s) REVERSE GENETICS Known Unknown Both very difficult in trees:
q  Size
q  Generation cycles
q  Outcrossing mating system
Genotype ‘BIG TREE GENE’ A ‘shortcut’ forward genetics - activation
tagging
Untransformed Genome
Transformed Genome
Transcriptional Enhancers
Transformation
Leaf Size Gene
Leaf Size Gen
Normal
Transcription
Hyperactivated
Transcription
Wild Type
Gain-of-Function Mutant
Populus Tagged Line
LB
pBstKS+ 4x35Se
RB
Purify
Sequence
…... Ecosystems Managed and Unmanaged Ecological implica@ons Bud dormancy and bud-­‐break Organisms Tissues/Organs Eco-­‐devo Cells Metabolites Proteins RNA Genes Regula4on of meristem Development Discovery of bud break ac4va4on tagged mutants FIELD Growth Chamber Posi4oning of the tag Ac4vated gene is homolog of DORNROSCHËN (DRN) = The Sleeping Beauty DORNROSCHËN (DRN) = The Sleeping Beauty
EBB1 is localized in the L1/L2 of Shoot Apical Meristem Expression of EBB1 during dormancy cycle EBB1
UBI4
0.8
0.7
Relative intensity
rrrrrrrrrrrrrrrrrrrrr
0.6
0.5
0.4
0.3
0.2
0.1
0
September
October
November
December
January
February
March
April
June
Transgenic manipula4on of EBB1 affects bud break 14
30
*
8
6
Days to bud-break
10
***
4
2
0
PRO35S:EBB1
Untransf ormed
Control
Transgenic Control
20
10
Genotype
0
30
WT 717-1B4
30
*
Days to bud-break
PRO35S::EBB1
Days to bud-break
Days to bud-break
12
20
10
WT-717
amiEBB1
*
20
10
0
WT-717
0
WT-717
amiEBB1
amiEBB1
Eco-­‐devo implica@ons Positive correlation for nonsynonymous SNP with
elevation
1.000
Frequency of A
0.900
r = 0.51, P = 0.042 0.800
0.700
r
0.600
0.500
0.400
1350
T-Square Test Statistic
8
1550
1750
1950
2150
2350
2550
Elevation (m)
6
4
2
0
1800
2000
2200
2400
2600
Sequence Position
2800
3000
Two loci (2119 and 2127) that showed an associa@on with bud flush (P = 0.007 for 2119 and P=0.09 for 2127) in a common garden experiment …... Ecosystems Managed and Unmanaged Organisms Tissues/Organs Cells Metabolites Proteins RNA Genes Evolu@onary and biotechnological implica@ons Wood anatomy in adapta4on and evolu4on of plants Increased wood produc4on Evo-­‐devo Biotechnology Regula4on of secondary growth Development Wood development
Cambium Zone Secondary Phloem
BARK
Daughter cells
Cambium initials
Daughter cells
Differentiation into phloem
Developmental boundary
Pluripotency
Developmental boundary
Differentiation into xylem
Secondary Xylem
WOOD
Discovery of poplar activation tagged line with
increased secondary growth
Mutant Yordan Yordanov
WT Positioning and validation of activation
A
A
LG_X
LG_X
LBD11
B
B
Blue-PPR
WT Mutant Ubiquitin
Cyclophilin
LG_X
PtaLBD11
WT Mutant Blue-PP
LG_X:18757850-18760033
53 grail3.0010003901
90
Cloning the gene
AT3G11090|LBD21
AT1G65620| AS2|LBD6
grail3.0010011101
grail3.0022002901
14
AT5G66870|LBD36
64
10
Ubiquitin
LG_X
grail3.0022010801
99
LG_X:18769015:18770840
C
gw1.VII.3089.1
98 gw1.V.3873.1
AT4G22700|LBD32
C
M
AT5G35900|LBD35
gw1.VIII.948.1
AT2G23660|LBD10
gw1.XII.1718.1
gw1.XV.1687.1
38
3
AT5G63090.1|LOB
AT3G27650|LBD25
grail3.0054008101
84
65 eugene3.00131258
gw1.XIX.2703.1
81
AT2G40470|LBD15
53
gw1.87.10.1
84
estExt f genesh4 pg.C LG X19 64 (PtaLBD1 )
AT2G30340|LBD13|
gw1.VIII.948.1
estExt fgenesh4 pg.C LG X1964
53
AT1G07900|LBD1
7
86
AT1G 0790 0|LBD1
AT2G28500|LBD11|
AT1G16530|LBD3
eugene3.00180176
gw1.VI.80.1
5
estExt fgenesh4 pg.C LG I0705
grail3.0018050401
58
eugene3.00070807
24
AT2G30130|LBD12
Class Ia
fgenesh4 pg.C LG I002068
50
16
37
86
AT1G31320|LBD4
fgenesh4 pm.C LG V000159
2
gw1.IX.2895.1
AT2G 2850 0|LBD11
See Suppl . Figur e 1
estExt fgenesh4 pg.C LG X1642
estExt Genewise1 v1.C LG VIII0894
gw1.XV.3141.1
96
gw1.XII.1626.1
36
AT3G26620|LBD23
5
99 AT3G26660|LBD24
gw1.X.3170.1
0.030
eugene3.00141147
gw1.XIV.812.1
90
46
0.025
0.020
0.015
0.010
0.005
0.000
gw1.II.1241.1
27
AT2G45410|LBD19
AT4G00210|LBD31
AT4G00220|LBD30
21
AT2G45420|LBD18|
33
gw1.II.1242.1
12
67 eugene3.00140160
eugene3.00051284
28
45
fgenesh4 pm.C LG II000194
71
gw1.64.470.1
51
AT5G06080|LBD33
18
66
fgenesh4 pg.C LG X001825
AT3G03760|LBD20
89
gw1.XIII.2161.1
AT2G31310|LBD14
22
AT2G42430|LBD16
21
AT2G42440|LBD17
30
AT3G58190|LBD29
78
91
gw1.II.1801.1
AT1G36000|LBD5
98
99
AT2G19510|LBD8
AT2G19820|LBD9
fgenesh4 pg.C scaffold 187000022
AT1G06280|LBD2
eugene3.00190839
91
eugene3.01420087
65
AT3G50510|LBD28
AT1G72980|LBD7
Class Ib
q  Homology to transcription factors of the LATERAL ORGAN
BOUNDARIES family
100 eugene3.01190031
24
90
eugene3.00440105
AT3G13850|LBD22
grail3.0005001801
67
q  Gene family comprised of 57 members in poplar
fgenesh4 pg.C LG XII000714
54
AT3G47870|LBD27
AT3G27940|LBD26
fgenesh4 pg.C LG XII000484
69 fgenesh4 pg.C LG IV000564
fgenesh4 pm.C scaffold 123000043
47
AT1G67100|LBD40
AT3G02550|LBD41
80
AT1G68510|LBD42|
95
gw1.X.210.1
59
65
gw1.VIII.1129.1
grail3.0001067101
fgenesh4 pm.C LG I001074
28
AT3G49940|LBD38
AT5G67420|LBD37
93
94 gw1.6210.2.1
26
33
gw1.II.3041.1
estExt fgenesh4 pg.C LG XIV0088
14
AT4G37540|LBD39
grail3.0089001701
72
estExt fgenesh4 pm.C LG V0239
0.500.450.400.350.300.250.200.150.100.050.00
Class II
q  Closest to LBD1 and LBD11 from Arabidopsis
LBD1 expression and localization
C
E
Xy
CZ
Ph
PhF
Relative Expression
A 1.5
1
0.5
0
A
L
PSt
SSt
Ph
Xy
R
q  Highest expression in
Phloem
q  Localized in a narrow region
including the cambium zone
q  Signal shifted to phloem side
Recapitulation via retransformation
Increased rays initiation and proliferation
53 grail3.0010003901
90
grail3.0022010801
AT3G11090|LBD21
AT1G65620| AS2|LBD6
99
grail3.0010011101
grail3.0022002901
14
AT5G66870|LBD36
64
10
gw1.VII.3089.1
98 gw1.V.3873.1
AT4G22700|LBD32
AT5G35900|LBD35
AT2G23660|LBD10
gw1.XII.1718.1
gw1.XV.1687.1
38
3
AT5G63090.1|LOB
AT3G27650|LBD25
grail3.0054008101
65 eugene3.00131258
gw1.XIX.2703.1
81
AT2G30340|LBD13|
AT2G40470|LBD15
gw1.87.10.1
84
gw1.VIII.948.1
Poplar LBD genes are predominantly
expressed in secondary phloem and
xylem
estExt fgenesh4 pg.C LG X1964
53
AT1G07900|LBD1
7
86
AT2G28500|LBD11|
AT1G16530|LBD3
eugene3.00180176
estExt fgenesh4 pg.C LG I0705
grail3.0018050401
58
AT1G31320|LBD4
fgenesh4 pm.C LG V000159
2
eugene3.00070807
24
AT2G30130|LBD12
Class Ia
fgenesh4 pg.C LG I002068
50
16
37
gw1.IX.2895.1
estExt fgenesh4 pg.C LG X1642
estExt Genewise1 v1.C LG VIII0894
gw1.XV.3141.1
96
gw1.XII.1626.1
36
AT3G26620|LBD23
5
99 AT3G26660|LBD24
gw1.X.3170.1
eugene3.00141147
gw1.XIV.812.1
90
46
gw1.II.1241.1
27
AT2G45410|LBD19
AT4G00210|LBD31
AT4G00220|LBD30
21
AT2G45420|LBD18|
33
gw1.II.1242.1
12
67 eugene3.00140160
eugene3.00051284
28
45
Relative expression
A
gw1.VI.80.1
5
fgenesh4 pm.C LG II000194
71
1.5
q  Are there secondary
xylem-specific genes?
0.5
0
PtaLBD1
PtaLBD1
AT5G06080|LBD33
18
66
SSt
1
gw1.64.470.1
51
PSt
fgenesh4 pg.C LG X001825
PtaLBD4
PtaLBD4
PtaLBD15
PtaLBD15
PtaLBD18
PtaLBD18
AT3G03760|LBD20
89
gw1.XIII.2161.1
AT2G31310|LBD14
22
AT2G42430|LBD16
21
B2
AT2G42440|LBD17
30
AT3G58190|LBD29
78
91
gw1.II.1801.1
AT1G36000|LBD5
98
99
Xylem
Phloem
fgenesh4 pg.C scaffold 187000022
AT1G06280|LBD2
eugene3.00190839
91
eugene3.01420087
65
AT3G50510|LBD28
AT1G72980|LBD7
Class Ib
100 eugene3.01190031
24
90
eugene3.00440105
AT3G13850|LBD22
grail3.0005001801
67
fgenesh4 pg.C LG XII000714
54
AT3G47870|LBD27
AT3G27940|LBD26
fgenesh4 pg.C LG XII000484
69 fgenesh4 pg.C LG IV000564
fgenesh4 pm.C scaffold 123000043
47
AT1G67100|LBD40
AT3G02550|LBD41
80
AT1G68510|LBD42|
95
gw1.X.210.1
59
65
gw1.VIII.1129.1
grail3.0001067101
fgenesh4 pm.C LG I001074
28
AT3G49940|LBD38
AT5G67420|LBD37
93
94 gw1.6210.2.1
26
33
gw1.II.3041.1
estExt fgenesh4 pg.C LG XIV0088
14
AT4G37540|LBD39
grail3.0089001701
72
estExt fgenesh4 pm.C LG V0239
0.500.450.400.350.300.250.200.150.100.050.00
Class II
Relative expression
AT2G19510|LBD8
AT2G19820|LBD9
q  Vascular cambium is
bifacial in poplar and
many other trees
q  LBD15 and LDB18
are expressed
predominantly in
secondary xylem
1
0
PtaLBD1
PtaLBD1
PtaLBD4
PtaLBD4
PtaLBD15
PtaLBD15
PtaLBD18
PtaLBD18
q  Almost perfect division
of labor – 2 phloem
and 2 xylem-specific
genes
Expression of meristem and phloem identity
genes is affected in the mutant
Meristem Iden4ty Gene Phloem Iden4ty Gene PtaLBD1 is down-regulated by auxin
Relative expression
A
2
Untreated
q  Auxin downregulates the
gene
Auxin treated
q  Several auxin signaling
genes are co-regulated
with LBD1 and may be
upstream regulatory
factors
1
0
PtaLBD1
15
PtaARF1
PtaARF2
PtaIAA8
PtaLBD1
PtaARF19
10
5
oo
ts
R
Xy
lem
ar
y
st
Se
em
co
nd
ar
y
ste
m
le
af
Pr
im
M
at
ur
e
Yo
un
g
lea
f
0
Ap
ex
Expression (log2 scale)
B
PtaPIN1
Model of LBD regulatory role
Auxin
Xylem
Other
unknown
gene targets
Boundary
LBD18 LBD15
Cambium Zone
ARK1
Other unknown
gene targets
Boundary
LBD1 LBD4
Phloem
APL
Other
unknown
gene
target
q  Involvement of auxin in defining the boundary
q  Dual roles of LBD in suppressing meristem and activation of
proliferation/differentiation genes
q  USDA grant to further investigate the hypothesis raised by
this model
Expansion of LBD1 in
Vitis corresponds to
specifics of its wood
anatomy
B
89
77
Vitis|003803
A
Vitis
B
Rays
Vitis|012976
WT-717
Vitis|018542
17
Vitis|035342
20
Populus| gw1.VIII.948.1
Populus| LBD1
Arabidopsis| AT1G07900
72
Arabidopsis| AT2G28500
11
Vitis|012309
75
Rays
2
Vitis|045864
Vitis|030340
2
Vitis|043817
Vitis|005837
PtaLBD1-oe
19
Vitis|025331
26
Vitis|013011
95
0.08
0.06
0.04
0.02
Vitis|031466
0.00
and LBD1 homologs. (A) Wood anatomy ultiseriate rays in Vitis (from Rays
Evo-devo of wood formation
q  Role of wood anatomy in ecological adaptation and
thus plant evolution has been long discussed but
the underlying genes and molecular mechanisms
largely unknown
q  Changes in the number (and/or expression) of
these genes through plant evolution may have
provided structural and functional evolutionary
innovations in wood anatomy in relation to species
growth habit and biology
q  The multiseriate rays and increased secondary
phloem production in Vitis resembles the poplar
transgenics with increased PtLBD1 expression.
Thus the putative increased LBD1 gene dosage in
Vitis corresponds to its wood anatomical features.
q  Working with Dr. Oliver Gailing on expanding these
findings across more taxa and correlating the
woody habit with sequence-derived phylogenies for
the LBD genes
Biotechnology •  Patent applica@on is pending on the four genes •  Working with Dr. Oliver Gailing and scien@sts from the Oakridge DOE na@onal laboratory on associa@on studies that can iden@fy polymorphisms in the 4 genes that may help breeders in early selec@on for increased wood produc@on •  Preliminary results show that upregula@on of the xylem-­‐specific genes leads to increased xylem produc@on. Tools and approaches Proteomics OMICS Metabolomics Transcriptomics Other ‘omics’ Genomics Modeling and inferences Valida@on System knowledge Advanced breeding and management prac@ces Gene@cs Lignocellulosic bioenergy crops will be primarily grown on marginal lands Pictures courtesy Brian Stanton – Greenwood Resources
Marginal lands Intensive poplar planta@ons in the Pacific Northwest of the US The hidden part Nutrients Water Regula4on of poplar root architecture in response to nitrogen TRANSCRIPTOMIC APPROACH FORWARD GENETICS APPROACH Root treatments –(-­‐N) Ac4va4on tagging popula4on – 5,000 lines Microarrays Bioinforma4c Analysis Screening under (-­‐N and –H2O) Mutants Project Web Site hDp://treesbio.com/ Iden4fica4on of candidate genes Key regulatory genes Transgenics of key regulatory genes Phenotypic analyses Recapitula4ons via retransforma4on Nitrogen – a solu4on and problem 1860 1990 hcp://home.iprimus.com.au/nielsens/nkiller.html#ref Poor nitrogen u4liza4on efficiency •
•
•
•
30-­‐70% of the applied N is NOT u@lized by the crop plants Result of decades of breeding under luxurious N levels Current prac@ces unsustainable environmentally and economically The excess N •  Contaminates water •  Contribute to greenhouse gas emissions •  Increases incidence of disease vectors hcp://www.scien@ficamerican.com/ar@cle.cfm?id=algal-­‐blooms-­‐may-­‐
become-­‐the-­‐norm-­‐in-­‐lake-­‐erie Experimental design Root measurements 0h
Treatment
Control Basal media 50mM KNO3 Affy
Low N 0.05mM KNO3
Affy
6h
12h
24h
48h
96h
504h
Affy
Affy
Affy
Affy
Affy
Affy
Affy
Affy
Affy
Affy
Affy
Affy
Sta4s4cal analysis Modeling Gene4c Networks Nitrogen depriva4on promotes poplar root prolifera4on and elonga4on 50
Main
root
lengthroot
(cm)
Lateral
roots/main
50
50
Low N
Control
40
35
12
12
**
Elonga4on 30
25
88
**
20
**
15
44
**
10
Lateral roots/main root
16
16
45
40
40
**
Lateral root prolifera4on 30
30
20
20
10
10
*
5
Control Medium
Total lateral roots length/main root
Low Nitrogen
0h0
0h
1d
24
1d
40
40
**
30
30
20
20
10
10
0
0
**
0
0h
241d
00
2d
4d
3w
48
96
2d
4d 504h
3w
2d
48
**
4d
96
3w
504h
0
0h
80
80
Roots dry biomass (mg/plant)
0
00
241d
482d
4d
96
Biomass accumula4on 3w
504h
**
60
60
40
40
20
20
0
0
0
0h
1d
24
2d
48
4d
96
3w
504h
Massive global changes in the root transcriptome in response to N deficiency Time 6h 12h 24h 48h 96h 504h Total DEGs 3704 1883 1309 3808 2483 3328 Downregulated 2194 740 506 1721 1154 1380 Upregulated 1510 1143 803 2087 1329 1948 Time BP MF CC Total Percentage 6h 301 63 46 410 25.3% 12h 125 29 4 158 9.7% 24h 112 26 13 151 9.3% 48h 260 35 19 314 19.4% 96h 243 23 43 309 19.0% 504h 200 48 33 281 17.3% •
•
•
•
9,198 differen@ally expressed genes More than 1,500 GO terms were enriched All @me points reported highly significant changes Slight dip at 12h and 24h Common temporal responses •  28 biological processes are common for all 4me points •  More than half (15/28) of these processes belong to Response to s4mulus – both endogenous and exogenous •  21/28 processes also enriched in Arabidopsis – interspecific core set? Metabolic Processes ( 5 )
GO:0019748 organic cyclic compound
biosynthetic process
GO:0044281 secondary metabolic process
GO:0008152
GO:0055114
GO:0044281
Average Fold Change
Description
GO Tree
6
2
0
-2
Response to Stimulus (15 )
GO:0042221 response to chemical stimulus
24
48
96 504 h
5.07E-26
1.21E-51
4.04E-54
small molecule metabolic process
oxidation-reduction process
GO:0050896
12
p-value
4.31E-42
2
0
-2
1.78E-69
GO:0009628
response to abiotic stimulus
8.22E-24
GO:0009719
response to endogenous stimulus
1.08E-16
GO:0006950
response to stress
4.66E-3
GO:0051716
cellular response to stimulus
5.87E-23
GO:0006955
immune response
1.63E-30
GO:0009607
response to biotic stimulus
1.29E-23
GO:0051179
GO:0051234
GO:0006810
Localization ( 8 )
establishment of localization
transport
2
0
-2
1.46E-33
4.74E-33
Temporal changes in the root response to LN GO #
GO-term
Average Fold Change (AFC)
NG p-value
Signaling and signal transduction
GO:0023052
GO:0044700
GO:0007165
GO:0009755
GO:0009867
signaling
single organism signaling
signal transduction
hormone-mediated signaling
jasmonic acid mediated signaling
141
141
141
62
45
2.35E-09
2.35E-09
2.35E-09
1.08E-05
7.95E-09
103
63
46
46
497
155
148
76
192
96
55
147
509
363
491
509
404
230
307
165
136
9.97E-05
2.26E-04
5.79E-04
2.62E-04
2.32E-08
6.46E-08
8.46E-11
5.99E-05
9.25E-06
1.50E-05
8.03E-05
1.54E-10
8.12E-08
6.23E-13
3.41E-18
1.31E-07
1.92E-11
1.78E-11
9.16E-12
5.94E-07
2.31E-08
95
56
73
65
65
42
56
59
56
82
1.75E-06
1.64E-06
9.84E-06
6.93E-06
1.10E-06
1.12E-05
1.09E-04
6.00E-05
0.000109
0.000848
Growth & development
GO:0048589
GO:0040008
GO:0035266
GO:0010075
GO:0007275
GO:0050793
GO:0051239
GO:2000241
GO:0009653
GO:0048580
GO:0009909
GO:2000026
GO:0044707
GO:0048856
GO:0071841
GO:0032501
GO:0032502
GO:0048513
GO:0048731
GO:0009888
GO:0040007
developmental growth
regulation of growth
meristem growth
regulation of meristem growth
multicellular organismal development
regulation of developmental process
regulation of multicellular organismal
regulation of reproductive process
anatomical structure morphogenesis
regulation of post-embryonic development
regulation of flower development
multicellular organismal development
single-multicellular organism process
anatomical structure development
biogenesis at cellular level
multicellular organismal process
developmental process
organ development
system development
tissue development
growth
Root development
GO:0022622 root system development
GO:0048364 root development
GO:0010015 root morphogenesis
GO:0010053 root epidermal cell differentiation
GO:0010054 trichoblast differentiation
GO:0048767 root hair elongation
GO:0048765 root hair cell differentiation
GO:0021700 developmental maturation
GO:0048764 trichoblast maturation
GO:0060560 developmental growth in morphogenesis
-3
3
Fold change scale
6
12
24
48
96 504
The temporal trends in GO term enrichment corresponded very well with the observed root morphological responses under LN: •  6h – 24 h Signaling ontologies •
48 h-­‐96 h Growth and development •
504 h Root development Func4onal and system inferences vs. cataloging Gene@c network analysis allow inferences about the connec@vity, organiza@on and hierarchy of the network. Sta@s@cal methods cannot discern direct interac@ons from cascading effects. ga gb a b c gc ga,gb gc,gd ge gd
d ge e Response to nitrogen deficiency involves hierarchically-­‐structured sub-­‐networks •
Super hubs are regulatory factors that exclusively connect hub genes •
Super hubs connected form 5 to 10 hub genes •
Each hub gene connected to 100s of genes •
Resembles a hierarchical regulatory structure •
High connec@vity as some of the hub genes shared connec@ons with the super hubs Super hubs Shared Hubs Hubs PtaNAC1 super hub networks PtaNAC1
GASA1
Regulatory module
Gibberellins Cell elonga@on GASA1-linked
(110 Genes)
EXPA17-linked
(157 Genes)
PPCK1-linked
(130 Genes)
WAK-linked
(115 Genes)
Nitrogen NIA2-linked
(162 Genes)
assimila@on RLK1-linked
(101 Genes)
AIR3-linked
(44 Genes)
EXPA17 PPCK1 WAK
NIA2
RLK1
PK
GO Ontology
GO:0010033 response to organic substance
GO:0031326 regulation of cellular biosynthetic process
GO:0009889 regulation of biosynthetic process
GO:0031323 regulation of cellular metabolic process
GO:0019222 regulation of metabolic process
GO:0009719 response to endogenous stimulus
GO:0008152 metabolic process
GO:0044710 single-organism metabolic process
GO:0044249 cellular biosynthetic process
GO:0006091 generation of precursor metabolites and energy
GO:0009058 biosynthetic process
GO:0044237 cellular metabolic process
GO:0006950 response to stress
GO:0050896 response to stimulus
GO:0008152 metabolic process
GO:0010468 regulation of gene expression
GO:0044710 single-organism metabolic process
GO:0060255 regulation of macromolecule metabolic process
GO:0050896 response to stimulus
GO:0006952 defense response
GO:0006950 response to stress
GO:0010033 response to organic substance
GO:0002376 immune system process
GO:0051707 response to other organism
GO:0006950 response to stress
GO:0050896 response to stimulus
GO:0009987 cellular process
GO:0051716 cellular response to stimulus
GO:0010035 response to inorganic substance
GO:0042221 response to chemical stimulus
GO:0006952 defense response
GO:0009627 systemic acquired resistance
GO:0051707 response to other organism
GO:0009697 salicylic acid biosynthetic process
GO:0009607 response to biotic stimulus
GO:0009696 salicylic acid metabolic process
GO:0006950 response to stress
GO:0006952 defense response
GO:0051704 multi-organism process
Super hub AIR3
Hubs Corrected p-values
2.51E-04
5.12E-04
5.23E-04
5.69E-04
7.55E-04
8.47E-04
3.01E-06
4.06E-05
4.25E-05
9.41E-05
1.28E-04
2.04E-04
6.93E-06
9.36E-05
2.45E-04
3.42E-04
6.11E-04
8.47E-04
7.32E-06
8.59E-06
4.08E-05
4.84E-04
1.16E-03
1.16E-03
1.12E-04
1.92E-04
7.21E-04
1.01E-03
1.15E-03
1.38E-03
7.88E-05
1.14E-04
1.17E-04
1.28E-04
1.35E-04
1.67E-04
5.39E-03
4.36E-03
7.55E-03
•
Large regulatory context of the network spanning from growth and hormones to nitrogen assimila@on •
18 of the enriched ontologies were among the set of 28 core ontologies that were enriched in all @me points T35S Root-­‐specific promoter •  Slight and insignificant differences in root growth and development under normal N levels •  Highly significant posi4ve changes under low N condi4ons Total LR length (cm/plant)
PtaNAC1 WT
ET304-PtaNAC1
6
5
*
4
3
2
1
0
30
*
25
20
15
10
5
0
25
Total LR number/plant
ET-­‐304 Root dry biomass (mg/plant)
PtaNAC1 upregula4on in poplar transgenics increased root growth specifically under LN *
20
15
10
5
0
Normal N Low N PtaNAC1
GASA1
WT
AIR3
*
Regulatory module
GASA1-linked
(110 Genes)
EXPA17
Relative expression
NIA2
RLK1
PK
AIR3
NIA2
*
•
PPCK1
WAKL
•
PPCK1-linked
(130 Genes)
WAK-linked
(115 Genes)
GASA1
*
•
NIA2-linked
(162 Genes)
*
*
RLK1-linked
(101 Genes)
PK
RLK1
•
AIR3-linked
(44 Genes)
*
*
LN
Control
LN
GO Ontology
GO:0010033 response to organic substance
GO:0031326 regulation of cellular biosynthetic process
GO:0009889 regulation of biosynthetic process
GO:0031323 regulation of cellular metabolic process
GO:0019222 regulation of metabolic process
GO:0009719 response to endogenous stimulus
GO:0008152 metabolic process
GO:0044710 single-organism metabolic process
GO:0044249 cellular biosynthetic process
GO:0006091 generation of precursor metabolites and energy
GO:0009058 biosynthetic process
GO:0044237 cellular metabolic process
GO:0006950 response to stress
GO:0050896 response to stimulus
GO:0008152 metabolic process
GO:0010468 regulation of gene expression
GO:0044710 single-organism metabolic process
GO:0060255 regulation of macromolecule metabolic process
GO:0050896 response to stimulus
GO:0006952 defense response
GO:0006950 response to stress
GO:0010033 response to organic substance
GO:0002376 immune system process
GO:0051707 response to other organism
GO:0006950 response to stress
GO:0050896 response to stimulus
GO:0009987 cellular process
GO:0051716 cellular response to stimulus
GO:0010035 response to inorganic substance
GO:0042221 response to chemical stimulus
GO:0006952 defense response
GO:0009627 systemic acquired resistance
GO:0051707 response to other organism
GO:0009697 salicylic acid biosynthetic process
GO:0009607 response to biotic stimulus
GO:0009696 salicylic acid metabolic process
GO:0006950 response to stress
GO:0006952 defense response
GO:0051704 multi-organism process
Corrected p-values
2.51E-04
5.12E-04
5.23E-04
5.69E-04
7.55E-04
8.47E-04
3.01E-06
4.06E-05
4.25E-05
9.41E-05
1.28E-04
2.04E-04
6.93E-06
9.36E-05
2.45E-04
3.42E-04
6.11E-04
8.47E-04
7.32E-06
8.59E-06
4.08E-05
4.84E-04
1.16E-03
1.16E-03
1.12E-04
1.92E-04
7.21E-04
1.01E-03
1.15E-03
1.38E-03
7.88E-05
1.14E-04
1.17E-04
1.28E-04
1.35E-04
1.67E-04
5.39E-03
4.36E-03
7.55E-03
2 of the 8 hubs were significantly expressed between WT and PtaNAC1 transgenics EXPA17-linked
(157 Genes)
*
Control
EXPA17 PPCK1 WAK
ET304-PtaNAC1
6 of the 8 hubs were differen@ally expressed in PtaNAC1 transgenics under low N treatment Some of the hubs like GASA1 completely reversed expression in the transgenics when compared to WT plants Expanding the work into the other super-­‐
hubs – appears that we are finding an unknown regulatory mechanism involved in the response to LN The Road Ahead …... Ecosystems Managed and Unmanaged Organisms •  Wood Biotechnology •  Evolu4on of woodiness •  Wood anatomy as evolu4onary adap4ve trait •  Root architecture and nitrogen and drought stress Tissues/Organs Cells Metabolites Proteins RNA Genes LOTS of work to be done here •  Role of LBD genes in regula4on of wood development •  Func4on gene discovery of genes affec4ng wood yield and quality •  System biology approach to understand root response to nitrogen and drought Aspen flushing in the
Canadian Rockies
GreenWood Resources
poplar plantations