1
2
3
4
Figure S1. Experimental design. Winter wheat genotypes (Geno 1- Ventnor and Geno 2- Karl
5
92) were grown at optimum temperatures (OT) until the onset of flowering. Thereafter, plants
6
were exposed to high night (HN), high day (HD), high day and night (HDN), or optimum
7
temperatures for 12 days. Each genotype had five plants (five biological replications) in each
8
temperature regime. Leaf samples for lipid extraction were collected from each plant on days 1,
9
6, and 12 during the stress period and four days after the stress was relieved (day 16). The
10
experiment was conducted two times (Run 1 and 2). GC- growth chamber.
1
1
2
Figure S2. Dendrogram showing the correlation among lipids included in groups in either
3
Ventnor or Karl 92. This dendrogram was produced using pooled data across genotypes. Lipids
4
were clustered using a single-linkage hierarchical algorithm based on Spearman’s correlation
5
coefficient (ρ). Thirteen clusters with ρ ≥ 0.80 are indicated by red and blue bars on the
6
dendrogram (two colors were used to easily distinguish the adjacent clusters). These clusters
7
represent co-occurring lipid groups and are labeled as Groups 1-13. In Group 9, MGDG(x/18:3)
2
1
indicates MGDG(18:4-O/18:3) or MGDG(19:3/18:3); MGDG(x2/y2) indicates MGDG(18:3-
2
2O/16:3), MGDG(18:4-O/17:1), or MGDG(19:3/17:1); and MGDG(x3/y3) indicates
3
MGDG(18:4-O/18:1), MGDG(19:3/16:3-2O), MGDG(18:3-2O/17:3), or MGDG(18:3-2O/16:4-
4
O).
3
6
A
PS(38:3)
B
PS(38:2)
C
PC(33:3)
D
PE(34:3)
E
PE(42:2)
F
MGDG(36:5)
G
DGMG(18:3)
3
Karl 92
Ventnor
Karl 92
Ventnor
Karl 92
Ventnor
Karl 92
Ventnor
0
-3
5
3
1
-1
-3
5
3
1
-1
4
2
0
-2
5
2
-1
1
-1
3
1
-1
H
MGDG(36:6)
3
0
-3
7
5
3
1
-1
7
5
3
1
-1
5
I
PC(18:3/18:3-2O)
J
TAG(16:0/34:3)
K
TAG(18:3/36:6)
3
1
-1
5
L
Sitosterol-Glc(16:0)
3
1
-1
7
M
Sitosterol-Glc
4
1
-2
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0
41
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Autoscaled lipid level
3
OT (25/15°C)
HN (25/24°C)
HD (35/15°C)
HDN (35/24°C)
1
4
1
Figure S3. Autoscaled levels of representative lipid analytes in individual plants. Thirteen lipids
2
presented in the figure represent the 13 different lipid groups. Each temperature regime
3
(optimum temperature [OT], high night temperature [HN], high day temperature [HD], and high
4
day and night temperature [HDN]) includes 40 plants out of which plants 1-10 were sampled on
5
day 1, plants 11-20 were sampled on day 6, plants 21-30 were sampled on day 12, and plants 31-
6
40 were sampled on day 16 (day 1 indicates the 1st day of stress and day 16 indicates the
7
recovery period, as stress was imposed for 12 days). While some lipids clearly change in
8
response to treatment, such as the increases in PC(33:3) (Panel C) or MGDG(36:5) (Panel F) or
9
decreases in MGDG(36:6) (Panel H) during heat treatment, there are also variations among
10
individual plants. These are sometimes as large as the variations in levels due to treatment. All
11
types of variations in lipid levels, including those due to treatment, to intrinsic differences in the
12
plants, or to the way that individual plants experience the treatment, reflect the levels of activities
13
of enzymes in the plants. Correlations of levels of various lipids (those in the same groups in
14
Figures 1, 2, and 3) across plants thus occur when lipids are formed by the same pathway.
5
Ventnor
Karl 92
PS(38:3)
0.026
Lipid/ Extracted dry mass (normalized mass spectral/mg)
0.021
PS(38:3)
0.026
OT
HN
HD
HDN
0.021
0.016
0.016
0.011
0.011
0.006
0.17
0.006
0.17
PS(40:3)
PS(40:3)
0.13
0.13
0.09
0.09
0.05
0.05
PS(38:2)
0.017
0.013
0.013
0.009
0.009
0.005
0.005
0.14
PS(38:2)
0.017
0.14
PS(40:2)
PS(40:2)
0.11
0.11
0.08
0.08
0.05
0.05
0
2
4
6
8
10
12
14
16
0
18
2
4
6
8
10
12
14
16
Days
1
2
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
3
next page). (A) Lipid Group 1 (Phosphatidylserine [PS] with an 18:3 acyl chain and a long acyl
4
chain) and Lipid Group 2 (PS with an 18:2 acyl chain and a long acyl chain).
6
18
Karl 92
Lipid/ Extracted dry mass (normalized mass spectral signal/mg)
0.04
Ventnor
0.04
OT
HN
HD
HDN
PE(33:3)
0.03
0.03
0.02
0.02
0.01
0.01
PC(33:3)
0.17
PE(33:3)
PC(33:3)
0.17
0.13
0.13
0.09
0.09
0.05
0.05
0.01
0.05
0.01
0.05
PI(33:3)
PI(33:3)
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
0.04
0.04
PI(35:3)
PI(35:3)
0.03
0.03
0.02
0.02
0.01
0.01
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
1
2
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
3
next page). (B) Lipid Group 3- Extraplastidic lipids with a 15:0 or 17:0 acyl chain combined
4
with an 18:3 acyl chain.
7
Karl 92
Ventnor
OT
HN
HD
HDN
LPE(18:3)
0.025
0.019
0.019
0.013
0.013
0.007
0.14
0.007
0.14
DAG(18:3/18:3)
0.10
0.10
0.06
0.06
0.02
0.02
PE(34:3)
0.7
1
2
DAG(18:3/18:3)
3
4
5
6
PE(34:3)
0.7
0.5
0.5
7
0.3
0.3
8
0.45
Lipid/Extracted dry mass (Normalized mass spectral signal/mg)
LPE(18:3)
0.025
0.45
PE(36:6)
0.35
0.35
0.25
0.25
0.15
0.15
0.05
0.05
0.95
10
11
0.95
PE(36:5)
9
PE(36:6)
12
PE(36:5)
13
0.75
0.75
0.55
14
0.55
0.35
15
0.35
PC(34:3)
5.1
PC(34:3)
5.1
4.4
4.4
3.7
3.7
3.0
3.0
16
17
18
2.3
2.3
PC(36:6)
2.6
2.6
2.1
2.1
1.6
1.6
1.1
1.1
0.6
0.6
PI(34:3)
2.6
2.1
1.6
1.6
1.1
1.1
0.6
0.6
PI(36:6)
19
20
21
PI(34:3)
2.6
2.1
0.13
PC(36:6)
22
23
24
25
PI(36:6)
0.13
0.09
0.09
26
0.05
0.05
27
0.01
0.01
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
8
1
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
2
next page). (C) Lipid Group 4- Extraplastidic lipids with an 18:3 acyl chain and an 18:3, 18:2 or
3
16:0 acyl chain.
9
Karl 92
Ventnor
1.0
1.0
PE(34:2)
PE(34:2)
0.8
0.8
0.6
0.6
OT
HN
HD
0.4
0.7
0.7
0.5
0.5
0.3
0.3
0.017
0.017
PE(36:2)
0.013
0.013
0.009
0.009
0.005
0.005
PE(38:4)
0.017
0.012
0.007
0.007
0.002
0.002
PE(42:3)
0.012
0.007
0.007
0.002
0.002
0.027
0.027
0.019
0.019
0.011
0.011
0.003
0.003
2
4
6
8
10
PE(42:3)
PE(42:2)
0.035
PE(42:2)
0
PE(38:4)
0.017
0.012
0.035
PE(36:2)
0.017
0.012
0.017
PE(36:4)
0.9
PE(36:4)
0.9
Lipid/ Extracted dry mass (normalized mass spectral/mg)
0.4
HDN
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
1
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
2
next page). (D) Lipid Group 5- Extraplastidic lipids with an 18:2 acyl chain (continued on next
3
page).
10
Karl 92
Ventnor
PC(34:2)
4
3
3
OT
HN
HD
HDN
2
2
PC(36:5)
2.8
Lipid/ Extracted dry mass (normalized mass spectral/mg)
PC(34:2)
4
2.2
2.2
1.6
1.6
1.0
1.0
2.3
2.3
PC(36:4)
1.7
1.7
1.1
1.1
0.5
0.8
0.5
0.8
PC(36:3)
0.6
0.6
0.4
0.4
0.2
0.2
PC(36:4)
PC(36:3)
PC(36:2)
0.18
PC(36:2)
0.18
PC(36:5)
2.8
0.15
0.15
0.12
0.12
0.09
0.09
0.06
0.06
PC(38:5)
PC(38:5)
0.033
0.033
0.025
0.025
0.017
0.017
0.009
0.009
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
1
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
2
next page). (E) Lipid Group 5- Extraplastidic lipids with an 18:2 acyl chain (continued on next
3
page).
11
Karl 92
Ventnor
PI(34:2)
1.6
1.3
1.3
OT
HN
HD
HDN
1.0
Lipid/ Extracted dry mass (normalized mass spectral/mg)
PI(34:2)
1.6
0.7
1.0
0.7
PI(36:5)
0.12
PI(36:5)
0.12
0.09
0.09
0.06
0.06
0.03
0.03
PI(36:4)
PI(36:4)
0.075
0.075
0.045
0.045
0.015
0.015
PI(36:3)
0.036
PI(36:3)
0.036
0.028
0.028
0.020
0.020
0.012
0.012
0.018
0.018
PI(36:2)
PI(36:2)
0.014
0.014
0.010
0.010
0.006
0.006
0.002
0.002
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
Days
1
2
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
3
next page). (F) Lipid Group 5- Extraplastidic lipids with an 18:2 acyl chain.
12
18
Karl 92
Ventnor
DGDG(34:2)
1.2
0.8
0.8
0.4
0.4
DGDG(34:1)
0.48
Lipid/ Extracted dry mass (normalized mass spectral signal/mg)
DGDG(34:2)
1.2
OT
HN
HD
HDN
DGDG(34:1)
0.48
0.33
0.33
0.18
0.18
0.03
0.03
DGDG(36:5)
10
DGDG(36:5)
10
7
7
4
4
1
1
DGDG(36:4)
DGDG(36:4)
1.3
1.3
0.9
0.9
0.5
0.5
MGDG(34:2)
0.35
MGDG(34:2)
0.35
0.25
0.25
0.15
0.15
0.05
0.05
MGDG(34:1)
MGDG(34:1)
0.19
0.19
0.13
0.13
0.07
0.07
0.01
0.01
MGDG(36:5)
36
MGDG(36:5)
36
31
31
26
26
21
21
16
16
11
11
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
1
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
2
next page). (G) Lipid Group 6- Polar lipids with 18:1 and/or 18:2 acyl chains (continued on next
3
page).
13
Ventnor
Karl 92
7
OT
HN
HD
HDN
5
MGDG(36:4)
7
5
3
3
1
1
MGDG(36:3)
0.5
Lipid/ Extracted dry mass (normalized mass spectral signal/mg)
MGDG(36:4)
0.3
0.3
0.1
0.1
3.2
3.2
PG(34:3)
2.7
2.7
2.2
2.2
1.7
1.7
1.2
1.2
PG(34:1)
0.8
0.6
0.4
0.4
PE(33:2)
0.020
0.014
0.014
0.008
0.008
0.002
0.002
PE(34:1)
PE(33:2)
PE(34:1)
0.05
0.03
0.03
0.01
0.01
PC(33:2)
0.10
PG(34:1)
0.026
0.020
0.05
PG(34:3)
0.8
0.6
0.026
MGDG(36:3)
0.5
PC(33:2)
0.10
0.07
0.07
0.04
0.04
0.01
0.01
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
1
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
2
next page). (H) Lipid Group 6- Polar lipids with 18:1 and/or 18:2 acyl chains (continued on next
3
page).
14
Karl 92
OT
HN
HD
HDN
1.3
Lipid/ Extracted dry mass (normalized mass spectral signal/mg)
0.9
Ventnor
PC(34:1)
PC(34:1)
1.3
0.9
0.5
0.5
0.1
0.1
PI(838 u)
0.020
0.014
0.014
0.008
0.008
0.002
0.002
PI(34:1)
0.13
PI(838 u)
0.020
PI(34:1)
0.13
0.09
0.09
0.05
0.05
0.01
0.01
DAG(16:0/18:1)
DAG(16:0/18:1)
0.007
0.007
0.004
0.004
0.001
0.001
0.029
0.029
DAG(18:1/18:3)
0.022
0.022
0.015
0.015
0.008
0.008
0.001
DAG(18:1/18:3)
0.001
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
1
2
3
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
4
next page). (I) Lipid Group 6- Polar lipids with 18:1 and/or 18:2 acyl chains.
15
Karl 92
0.13
Ventnor
0.13
DGMG(18:3)
OT
HN
HD
HDN
0.10
0.07
0.04
0.01
0.01
MGMG(18:3)
Lipid/Extracted dry mass (normalized mass spectral signal/mg)
2
3
MGMG(18:3)
0.13
0.09
0.09
0.05
0.05
0.01
0.01
4
5
6
DGDG(38:6)
DGDG(38:6)
0.35
1
0.07
0.04
0.13
DGMG(18:3)
0.10
0.35
7
0.25
8
0.15
9
0.25
0.15
0.05
DGDG(36:6)
51
DGDG(36:6)
51
44
44
37
37
30
30
10
11
12
23
184
23
184
MGDG(36:6)
MGDG(36:6)
154
154
124
124
94
94
64
64
13
14
15
34
34
PG(34:4)
10
PG(34:4)
10
7
7
4
4
16
17
18
1.9
1.9
SQDG(34:3)
SQDG(34:3)
1.5
1.5
19
1.1
1.1
20
21
0.7
0.7
1.7
1.7
SQDG(36:6)
1.3
1.3
0.9
0.9
0.5
0.5
SQDG(36:6)
22
23
24
0.1
0.1
0
2
4
6
8
10
12
14
16
18
Days
0
2
4
6
8
10
12
14
16
18
25
26
27
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
28
next page). (J) Lipid Groups 7 and 8- Plastidic lipids with 18:3 acyl chains.
16
Karl 92
Ventnor
OT
HN
HD
HDN
MGDG(x/18:3)
0.16
0.12
MGDG(x/18:3)
0.16
0.12
0.08
0.08
0.04
0.04
Lipid/ Extracted dry mass (normalized mass spectral signal/mg)
MGDG(x2/y2)
MGDG(x2/y2)
0.0035
0.0035
0.0025
0.0025
0.0015
0.0015
0.0005
0.0005
0.008
0.008
MGDG(x3/y3)
0.006
0.006
0.004
0.004
0.002
0.002
MGDG(18:3/18:3-2O)
0.25
MGDG(x3/y3)
MGDG(18:3/18:3-2O)
0.25
0.21
0.20
0.17
0.15
0.13
0.10
0.09
0.05
0.017
0.05
0.017
PC(18:3/18:3-2O)
0.013
0.013
0.009
0.009
0.005
0.005
0.001
PC(18:3/18:3-2O)
0.001
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
Days
1
2
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
3
next page). (K) Lipid Group 9- Ox-lipids. MGDG(x/18:3) indicates MGDG(18:4-O/18:3) or
4
MGDG(19:3/18:3); MGDG(x2/y2) indicates MGDG(18:3-2O/16:3), MGDG(18:4-O/17:1) or
5
MGDG(19:3/17:1); and MGDG(x3/y3) indicates MGDG(18:4-O/18:1), MGDG(19:3/16:3-2O),
6
MGDG(18:3-2O/17:3) or MGDG(18:3-2O/16:4-O).
17
18
Karl 92
Ventnor
0.05
DAG(16:0/18:2)
DAG(16:0/18:2)
Lipid/Extracted dry mass (normalized mass spectral signal/mg)
0.04
0.03
0.02
OT
HN
HD
HDN
0.01
0.026
TAG(16:0/34:3)
TAG(16:0/34:3)
TAG(16:0/34:2)
TAG(16:0/34:2)
TAG(16:0/34:1)
TAG(16:0/34:1)
0.021
0.016
0.011
0.006
0.001
0.026
0.021
0.016
0.011
0.006
0.001
0.009
0.007
0.005
0.003
0.001
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
Days
1
2
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
3
next page). (L) Lipid Group 10- TAGs and DAGs with 16:0 acyl chain(s).
18
Karl 92
Lipid/Extracted dry mass (normalized mass spectral signal/mg)
0.5
Ventnor
TAG(18:3/36:6)
TAG(18:3/36:6)
OT
HN
0.4
HD
HDN
0.3
0.2
0.1
0.0
0.5
TAG(18:3/36:5)
TAG(18:3/36:5)
TAG(18:3/36:4)
TAG(18:3/36:4)
TAG(18:3/36:3)
TAG(18:3/36:3)
0.4
0.3
0.2
0.1
0.0
0.16
0.11
0.06
0.01
0.026
0.021
0.016
0.011
0.006
0.001
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
Days
1
2
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
3
next page). (M) Lipid group 11- TAGs with 18:3 acyl chain(s).
19
18
Lipid/ Extracted dry mass (normalized mass spectral signal/mg)
Karl 92
Ventnor
Campesterol-Glc(16:0)
0.04
Campesterol-Glc(16:0)
0.04
OT
HN
HD
HDN
0.03
0.02
0.03
0.02
0.01
0.01
Stigmasterol-Glc(16:0)
0.017
Stigmasterol-Glc(16:0)
0.017
0.013
0.013
0.009
0.009
0.005
0.005
Sitosterol-Glc(16:0)
Sitosterol-Glc(16:0)
0.11
0.11
0.07
0.07
0.03
0.03
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
Days
1
2
3
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups (continued on
4
next page). (N) Lipid Group 12- Acylated sterol glycosides.
20
18
Lipid/ Extracted dry mass (normalized mass spectral signal/mg)
Karl 92
0.65
Ventnor
0.65
Campesterol-Glc
OT
0.55
HN
HD
0.45
0.45
HDN
0.35
0.35
0.25
0.25
0.15
0.15
Stigmasterol-Glc
0.38
Campesterol-Glc
0.55
Stigmasterol-Glc
0.38
0.30
0.30
0.22
0.22
0.14
0.14
0.06
0.06
2.5
2.5
Sitosterol-Glc
Sitosterol-Glc
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
Days
1
2
Figure S4. The kinetic changes of individual lipid levels in the 13 lipid groups. (O) Lipid Group
3
13- Sterol glycosides.
21
18