Transcript Plant Gravitational and Space Genomics Tour

Gravity
Mechanical
0 2 5 15 30 60 min after reorientation
Transcriptional Regulation during
Gravitropism in Arabidopsis Root Tips
0 2 5 15 30 60 min after reorientation
Heike Winter Sederoff, Jeffery M. Kimbrough,
Raul Salinas-Mondragon and Christopher Brown
NSCORT-Plant Gravitational Genomics Group,
Dept. Botany and Kenan Institute, NC State University
NC State University
[email protected]
Clusters of temporal expression
Rapid, temporal changes in the abundance of specific transcripts
occur in response to gravity-stimulation and these transcript level
changes reveal clusters of coordinating signaling events that are
required for the tissue to make a commitment to differential growth.
We have carried out microarray analysis of Arabidopsis root tips after
gravistimulation (reorientation) and mechanical stress and monitored
transcript levels ca. 24,000 genes during the first hour after either
stimuli. We identified genes responding to either stimulus specifically
as well as genes shared by both signalling pathways. The temporal
pattern of transcription and transcript stability of those induced genes
are very distinct. We identified stress (gravity vs mechanical) specific
gene expression and isoform specific expression. The earliest changes
of transcript levels were seen already 2 min after the stimulus.
Preliminary results show that different clusters of genes exhibit
changes in mRNA abundance with specific patterns following the
time-course during the first hour after gravistimulation (Fig.2).
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xyloglucan endotransglycosylase
Figure 2: Temporal
changes of transcript
levels of a xyloglucan
endotransglycosylase
XET (At4g30270),
sucrose transporter
SUC2, and a lipidtransfer protein-like gene
(At2g18370) in
Arabidopsis root tips
after gravity-stimulation.
lipid transfer protein-like
fold change after reorientation
Abstract
sucrose transporter SUC2
4
3
2
1
The Approach
0
0
First Results
We are currently still analyzing the vast amount of data generated by
this method. To get an impression of the quality of our microarrays, we
carried out real-time PCR for eight genes shown to be up-regulated
during gravitropism (see Fig. 3). Scatter Plots (see Fig.1) showed
excellent correlation in overall gene expression between treatments.
30
min after reorientation
45
60
For specific transcripts, changes in the rates of transcription vs transcript
degradation lead to decreases of their steady- state levels (Fig. 3).
1.0
fold-change of transcript level
We are using the Arabidopsis root tip (7 day old dark-grown
seedlings) to analyze the changes in transcript levels in response to
gravistimulation using the Arabidopsis ATH1 GeneChip (Affymetrix),
containing more than 22,500 probe sets representing approximately
24,000 genes. For our time-course experiment, approximately 150 root
tips were harvested before (0 time point) and 2, 5, 15, 30, 60 min after
reorientation (135◦) by pouring RNAlater (Ambion) onto the plates and
cutting off the root tips (5 mm). Mechanical stress controls seedlings (0,
2, 5, 15, 30, 60 min) were moved horizontally for 5 sec without
changing their orientation towards the vector of gravity.
15
extensinA
RNA-binding protein-like
pectinesterase like
0.8
0.6
0.4
0.2
0.0
0
15
30
min after reorientation
45
60
Figure 3: Decrease of
transcript levels of an
extensinA
(CAA47807.1), RNA
binding protein-like
(gi:16974555), and
pectinesterase-like
(PATX:E31214555) in
Arabidopsis root tips
after gravitystimulation.
Isoform-specific and gravity-specific transcripts
Stress:
Gravity
Mechanical
Gravity (specific)
Mechanical (specific)
Both Stresses
similar response:
opposite response:
up
down
1414
1115
1058
788
149
235
110
168
324
48
total
1563
1350
1168
956
397
25
Table I: Summary of transcript levels responding to gravity and/or
mechanical stimulation by increased (up) or decreased (down) steadystate levels of transcripts.
Figure 6: Hierarchical cluster showing mechanical stress regulated transcripts
fold-change transcript levels (microarrays)
3000
gravistimulated
2500
signal intensity
We found about 1,563 genes were regulated by gravity. Of those, 1,414
showed significant increases in transcript abundance during
gravistimulation while 149 showed decreased levels. During mechanostimulation, 1,350 genes showed significant regulation, 1,115 were
increased, while 235 showed a decrease in transcript abundance.
Gravity-specific changes were detected for 1,168 transcripts, while 956
transcript showed changes in transcript abundance only after mechanostimulation. Especially interesting are those genes which show opposite
responses in both signaling networks (see Table I).
relative transcript level changes gravity/mechanical (real time PCR)
Figure 1. Scatter Plot: X-axis values (log 10 signal intensities) correspond to
transcript profile before gravity stimulation. Y-axis values correspond to transcript
profile of root tips harvested after a 2 minute reorientation (135◦) in the gravity
vector plane. Innermost parallel green lines flanking the scatter indicate boundary
for genes up or down regulated greater than 2 fold (yellow: not expressed, blue:
expressed under one condition; red: expressed genes). Each dot represents a unique
gene.
For some clusters of genes, we found very specific expression of individual
isoforms of gene families to expressed within less than 5 min after
gravistimulation. Calmodulin1 (CaM1) was upregulated within 2-5 min after
gravistimulation, while none of the other calmodulin isoforms showed
significant effects (Fig. 5). After mechano-stimulation, CaM1 transcript levels
were down-regulated (Fig.4). Similar kinetics and specificity was found for
the mu isoform of 14-3-3’s.
Figure 4: Transcript
4.0
1.6
levels of CaM1 in
CaM1
gravity (microarray)
3.5
Arabidopsis root tips after
mechanical stress (microarray)
gravistimulation
gravity/mechanical stress (real time PCR)
3.0
1.4
compared to transient
2.5
mechanical stress. Data
2.0
1.2
from microarray analysis
1.5
(n=2) and confirmed by
real-time PCR (with
1.0
1
Actin8 as reference).
0.5
Upregulation of CaM1
0.0
0.8
occurs within 5 min of
0
15
30
45
60
min after
gravistimulation.
reorientation
cam1
2000
1500
14-3-3 mu
1000
cam3
500
cam
0
0
15
min
30
45
60
Figure 5: Specific
isoforms of CaM and
14-3-3 respond to
gravistimulation.
Upregulation of CaM1
and 14-3-3 mu, occurred
within less than 5 min
and steady-state
transcript levels were
back to control levels
after 30 min.
Methods: Total RNA was extracted and purified over a Qiagen RNeasy
column, amplified, and labeled with biotin. 10 ug of biotin labeled aRNA
(amplified RNA) was hybridized to the GeneChip® Arabidopsis ATH1
Genome Array (Affymetrix, Santa Clara, CA) containing more than 22,500
probe sets.
Acknowledgements: This work was supported by NASA. We thank Nina
Allen, Wendy Boss, Eric Davies, Steve Huber, Gloria Muday, Imara
Perera, Dominique Robertson, and Ron Sederoff for stimulating
discussion and support.
Figure 7: Hierarchical cluster showing gravity regulated transcripts