High Nighttime Temperature Stress
In Field Grown Rice
Jigar
1Department
1
Desai ,
Lovely Mae
2
Lawas ,
S.V. Krishna
2,3
Jagadish ,
1
Doherty
Colleen J.
of Biochemistry, North Carolina State University 2International Rice Research Institute, 3Department of Agronomy, Kansas State University
Dawn +
17.5h
Background
Rice Grown under High Nighttime Temperatures (HNT) with Ceramic Heaters
Figure 1: Average daily minimum
temperatures are increasing faster
than
the
daily
maximum
temperature, reducing the daily
temperature range (DTR) that plants
are exposed to on a daily basis.
The predicted impacts of climate change have made
tolerance to high temperatures an important target for
crop breeders. However, the effects of high
temperatures on plants depends on when the stress
occurs. For example, a small temperature increase at
night (1°C) has a significant negative impact on rice
yield and quality [1]. Yet the molecular mechanisms
causing this sensitivity to high nighttime temperatures
(HNT) are not well understood. Uncovering the
pathways affected by HNT and understanding the
molecular effects will provide markers for selection and
potential strategies to mitigate the negative effects of
HNT on yields. Over the past 100 years, the daily
minimum temperature has increased faster than the
daily maximum temperature (Figure 1) and this trend is
predicted to continue [2]. This observation emphasizes
the importance of understanding the effects of HNT on
plants to maintain productivity in the face of a changing
climate.
900.00
HNT
Heat
Control
Yield (g/m2)
800.00
700.00
Figure 2: Rice yield in field condition treated with HNT with ceramic heaters (orange bar, heaters shown in left panel)
or control, untreated plants (blue bar). Using the ceramic heaters, temperature was increased by 2-2.5oC at night
time. HNT results in a significant reduction in total yield for the IR64 variety.
DE genes
• Does HNT disrupt timing of expression?
• Are certain temperature sensitive processes affected by HNT?
• Are there molecular markers that are phase specific to assist
breeders?
Functional Enrichment of DE Genes and Cycling Genes
Differentially Expressed GO Term Enrichment
Differentially Expressed at Each Time Point (Control vs HNT)
Dawn +
7h
Figure 3: Panicle tissue was collected during 50% flowering at
8 different time points. Harvested tissue was used for
transcriptome and metabolome analysis.
IR64
Figure 4: Check out the effects of HNT on your favorite rice gene.
Dawn +
3.5h
Dawn +
10.5h
500.00
Transcript data for the time course is
available online through R’s Shiny web
applications. The link and QR code are
above.
Dawn
Dusk
600.00
https://jigardesai.shinyapps.io/IRRI_IR64_timecourse
Dawn +
21h
Dawn +
14h
GO term
Description
Number in input list
Number in BG/Ref
p-value
FDR
GO:0051252
regulation of RNA metabolic process
68
760
0.00025
0.049
GO:0008152
metabolic process
521
8041
4.60E-05
0.049
GO:0006355
regulation of transcription, DNA-dependent
68
758
0.00023
0.049
GO:0006351
transcription, DNA-dependent
71
799
0.00022
0.049
GO:0032774
RNA biosynthetic process
71
801
0.00023
0.049
GO:0043687
post-translational protein modification
109
1365
0.00027
0.049
GO:0006464
protein modification process
118
1495
0.00023
0.049
Metabolic Process GO Term Gene Clustering
HNT
Control
900
800
700
Figure 10: Functional Enrichment of GO terms for differentially expressed genes at any time point. The top seven GO terms are listed. To examine the expression pattern
of the transcripts within each category, the input list for the GO category was clustered using K-means based on expression in the control samples. Before the genes
were clustered, they were first normalized by their level of expression, so they can be plotted on the same graph. The expression is plotted in Control and HNT. For the
GO metabolic process there are dramatic changes in the patterns for some of the clusters.
# of genes
600
500
400
300
200
100
GO Term Enrichment of Genes Cycling Only in Control
0
Dawn
3.5h
7h
10.5h
Dusk
14h
17.5h
21h
Figure 5: Differentially expressed genes between HNT treated and control plants at each time point.
Most changes occur at dawn and dusk, but the effects extend into other time points, including those
where HNT and control plants are in the same temperature (3.5h and 7h) indicating that the effects
extend beyond the period of increased temperature.
Correlation of Normalized Counts
Between Consecutive Time Points
DE Genes Timepoint Overlaps
Dawn and 3.5h overlap
3.5h and 7h overlap
GO term
Description
Number in input list
Number in BG/Ref
GO:0005739
mitochondrion
374
4190
GO:0009536
plastid
412
GO:0044429
mitochondrial part
GO Term enrichment of Genes Cycling Only in HNT
p-value
FDR
GO term
Description
Number in input list
Number in BG/Ref
2.40E-10 9.50E-08
GO:0043687
post-translational protein modification
138
1365
3.70E-06 0.0013
4949
7.70E-08 1.60E-05
GO:0006464
protein modification process
149
1495
3.00E-06 0.0013
29
154
2.30E-06 0.00031
GO:0006796
phosphate metabolic process
135
1327
3.50E-06 0.0013
GO:0034641 cellular nitrogen compound metabolic process
40
252
1.50E-06
0.0022
GO:0006793
phosphorus metabolic process
135
1327
3.50E-06 0.0013
GO:0030964
NADH dehydrogenase complex
6
7
6.10E-05
0.0041
GO:0006468
protein amino acid phosphorylation
117
1126
6.80E-06 0.002
GO:0045271
respiratory chain complex I
6
7
6.10E-05
0.0041
GO:0043412
macromolecule modification
150
1542
9.30E-06 0.0022
GO:0005840
ribosome
42
316
4.10E-05
0.0041
GO:0016310
phosphorylation
121
1217
3.00E-05 0.0061
7h and 10.5h overlap
10.5h and Dusk overlap
p-value
FDR
Dusk and 14h overlap
17.5h and 21h overlap
Mitochondrion GO Term Gene Clustering
Control
HNT
C_Dawn C_3.5 C_7 C_10.5 C_Dusk C_14
C_17.5 C_21
>2 Timepoints DE
HNT_Dawn 260 42 3 0 0 0 14 193
0
5
10
15
20
25
30
35
45 Figure 7: Correlation between consecutive time
HNT_3.5
6640 221
55 1 0 0 1 43
# of genes
points
HNT_7 12 54 324
148(ex:
48 Dawn
1 0 is 3the correlation between Dawn
Figure 6: Transcripts identified as
for control
(blue) and HNT treated
HNT_10.5 0 0 and
25 3.5h)
291 113
10 6 plants
0
differentially expressed atHNT_Dusk
more than0 0 plants
5 109
248 26
18 1 there is a high degree of
(red).
In HNT
one time point.
HNT_14 33 1 1correlation
29 273 around
263 343 Dusk.
40
HNT_17.5 16 0 0 2 23 33 123 10
HNT_21
159 1 0Transcripts
0 3 3 10 81
Cycling
Column1
Lost in HNT
Gained in HNT
24
5639
5699
1913
1937
Gained in HNT Period==24
Lost in HNT AMP > 1
1661
1642
Lost in HNT AMP > 2
1377
1435
Lost in HNT AMP > 10
425
486
Lost in HNT AMP > 20
177
187
17
17
Lost in HNT AMP > 200
9
6
Metabolites
Citrate
Pyruvate
pyruvate
A
Decreases in heat
citrate
Isocitrate
isocitrate
AM_HNT
PM_Control
PM_HNT
1.1
AM_Control
AM_HNT
PM_Control
1.0
0.9
1.2
1.1
AM_Control
C_10.5 C_Dusk
0
0
1
0
148
48
291
113
109
248
29
273
2
23
0
3
C_14
0
0
1
10
26
263
33
3
C_17.5
14
1
0
6
18
343
123
10
C_21
193
43
3
0
1
40
10
81
PM_HNT
Alpha-ketoglutarate
Higher in AM
HNT AM
Control PM
HNT PM
Control
PM/AM
(3)
3−methyl−2−oxobutyrate
Control
PM/AM
(6)
HNT
PM/AM
(3)
HNT
PM/AM
(6)
3−methyl−2−oxovalerate
ascorbate (Vitamin C)
1.8
1.4
1.4
1.4
PM_Control
PM_HNT
1.1
1.0
AM_HNT
0.6
0.5
1.0
0.6
1.0
AM_Control
0.8
0.8
1.5
0.6
1.2
1.0
1.0
0.8
1.2
1.3
1.2
1.0
3.0
1.4
3.5
succinate
1.6
Succinate
fumarate
1.2
Fumarate
PM_HNT
1.5
1.6
AM_Control
AM_HNT
PM_Control
PM_HNT
AM_Control
AM_HNT
PM_Control
PM_HNT
Figure 11: Metabolite levels of the TCA cycle. Metabolic profiling was performed on Control (Green) and HNT
(Orange/Red) samples at Dawn and Dusk. For the mature panicles (3rd) the samples were prepared from a
portion of the tissue used for the transcript analysis. This profiling detected many TCA cycle metabolites. For
most TCA metabolites, there is a significant difference between Control and HNT levels. Over the two time
points, most of the TCA metabolites seem to show more sensitivity to HNT at Dusk compared to Dawn.
Conclusion
As previously reported, yield decrease under HNT is significant. Molecularly, the pattern of expression is disrupted for many
transcripts, the most prominent differences are detectible at dawn and dusk time points. Importantly the time of day the plants are
sampled is important for identifying transcript and metabolite differences. Functional categorization of differentially expressed
transcripts and transcripts with alterations in their rhythmic pattern reveal changes in GO Terms associated with metabolism.
There is an enrichment to post translational changes in genes that cycle in HNT conditions. There is a connection between the
genes that lose cycling in HNT and level of TCA metabolites. The disruption of the TCA also fits the transcripts conclusion
because of the enrichment mitochondrion GO term and other related metabolic GO terms in the genes that lose cycling in HNT.
This disruption of the metabolites levels could a be one potential causal mechanism for the reduction in yield under HNT.
0.9
PM_Control
1.4
AM_HNT
1.2
AM_Control
2.5
C_7
3
55
324
25
5
1
0
0
PM_Control
alpha−ketoglutarate
2.0
HNT_Dawn
HNT_3.5
HNT_7
HNT_10.5
HNT_Dusk
HNT_14
HNT_17.5
HNT_21
C_3.5
42
221
54
0
0
1
0
1
AM_HNT
0.7
0.8
0.9
1.0
TCA Cycle
Increases
in heat
C_Dawn
260
66
12
0
0
33
16
159
Control AM
PM_HNT
Higher in PM
0.6
Figure 9: Of the transcripts that are
cycling in both conditions (3788 genes)
many change their phase of max
expression in HNT. This Circos plot
shows the change in the time of max
expression of each transcript from
control plants (C, right side) to HNT
plants (HNT, left side). The table below
shows the number of genes changing
in each condition.
AM_Control
malate
1.3
Malate
0.4
0.6
1.2
0.8
0.6
1.3
1.0
1.4
0.8
1.2
1.5
1.0
1.4
Lost in HNT AMP > 100
Figure 8: Transcripts
with a cycling pattern
were identified using JTK
cycle [3] with a cutoff of
<.01 adjusted p-value.
About an equal number
of cycling genes are lost
or gained with HNT.
1.6
Lost in HNT Period==24
0.8
Cycling in HNT
0.6
Cycling in Control
# of Significant Compounds
Period
Post-translation Protein Modification GO Term Gene Clustering
Control
HNT
1.8
21 and Dawn Overlap
1.2
14h and 17.5h overlap
AM_Control
AM_HNT
PM_Control
PM_HNT
AM_Control
AM_HNT
PM_Control
PM_HNT
AM_Control
AM_HNT
PM_Control
PM_HNT
Figure 12: Metabolite Analysis of rice panicles grown in HNT or control
conditions. A) Total number of significantly different compounds for
each comparison for the third (3) or sixth (6) branch and examples of
metabolites with response to HNT detected differently depending on
time point sampled dawn (AM) or dusk (PM).
References
[1] Peng, S., J. Huang, J. E. Sheehy, R. C. Laza, R. M. Visperas, X. Zhong, G. S. Centeno, G. S. Khush, and K. G. Cassman.
"Rice Yields Decline with Higher Night Temperature from Global Warming." Proceedings of the National Academy of
Sciences 101.27 (2004): 9971-975.
[2] Easterling, D. R. "Maximum and Minimum Temperature Trends for the Globe." Science 277.5324 (1997): 364-67.
[3] HughesLab:JTK Cycle. (2015, October 12). OpenWetWare, . Retrieved 03:32, January 2, 2016
from http://openwetware.org/index.php?title=HughesLab:JTK_Cycle&oldid=907164.