2. Results
2.2 Genetic Network of Ascorbate Peroxidase Involving in Thermal-induced
2.2.2 Transcriptional Profiling of OgAPXOX Arabidopsis under Elevated Growth
To address this issue, we carried out an analyses of the expression profiling of
OgAPXOX and control plants in response to the elevated growth temperature by 3' IVT
Expression GeneChip (Affymetrix; Arabidopsis ATH1). All plants were growed under
LD-22°C condition. Until they had 6 leaves, a half of transgenic lines and wild type
would be cultivated under LD-30°C and sampled the leaves at 0 day under LD-30°C, at
1 day under LD-30°C and at 5 day under LD-30°C for further analysis. After
normalization of the expression level with wild type growing under 22°C by Gene
Spring GX10.5 (http://www.genomics.agilent.com/homepage.aspx), there were total of
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860 transcripts selected from the other treatments displaying up- or down- regulated
expression levels (>2 fold). The 860 transcripts were subsequently categorized into 10
clusters using k-means clustering in accordance with the pattern of their temporal
change in the expression profile (Fig. 21A). The clusters consisted of transcripts that
displayed overall trends of either increased (Fig. 21B, clusters 2-9) or decreased (Fig.
21B, cluster 1) gene expression over the different time points in the study. The detail of
all clusters is interpreted:
Cluster 1:
In Fig. 21B and Table. 1, total of 244 transcripts in cluster 1 displayed overall
tends of decreased gene expression. Gene-ontology (GO) analysis of genes in cluster 1 displayed the major subcategories in “molecular function” were function on DNA
binding and provided with transcription regulator activity and transcription factor activity. The major subcategory in “cellular component” was located at endomembrane
system. The major subcategories in “biological process” were related to transcription
factor activity, stress response and response to hormone stimulus. Noteworthily,
abundant genes in cluster 1 were predicted to encode various members of transcription
factors, including MYB, AP2 and protein in circadian clock. Three MYBs, AtMYB48
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(At3g46130), AtMYB3 (At1g22640), and AtMYB60 (At1g08810), in cluster 1 belong to
R2R3-MYB and have been characterized their function on anthocyanin biosynthesis.
Four genes encoding AP2 protein in cluster 1 belong to RAP2.4 subgroup which
mediating light and ethylene signaling, including At4g39780, At1g64380, At2g22200
and At1g74930. (Wang et al., 2008). Circadian clock genes, LHY (At1g01060) and
APRR9(At2g46790), regulated flowering time through the canonical CO-dependent
photoperiodic pathway (Nakamichi et al., 2007) and displayed drastically decreased
expression levels under elevated growth temperature. Therefore, it suggests that
antioxidant state (including anthocyanin and AsA), circadian rhythm and
phytohormones level would be altered in response to the change of growth temperature
and AsA state.
Cluster 2:
In Fig. 21B and Table. 2, total of 126 transcripts in cluster 2 displayed slightly
decreased expression pattern after 1 day under LD-30°C, but significant increase in
transgenic plants after 5 day under LD-30°C. GO analysis of genes in cluster 2 displayed the major subcategory in “molecular function” was function on nucleotide
binding. The major subcategory in “cellular component” was located at plasma
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membrane. The major subcategories in “biological process” were related to defense
response, phosphate metabolic process, response to organic substance and nitrogen
compound biosynthetic process. Noteworthily, abundant genes in cluster 2 were
predicted to encode various members of stress-related transcription factors, including
AtMYB70 (At2g23290) and AtWRKY11 (At4g31550). The transcription factor
AtWRKY11 acted as negative regulator for plant in susceptible to Pseudomonas syringae
pv tomato (Kroj et al., 2006). Moreover, AtMYB70 was likely to be associated with
stress responses but still unclear about its detail function. All of them displayed striking
increase in OgAPXOX under LD-30oC and suggests that their probable functions on
coordination of elevated growth temperature and sensing the redox state change caused
by OgAPXOX.
Cluster 3:
In Fig. 21B and Table. 3, cluster 3 contains 29 transcripts that displayed
significantly decreased expression pattern after 1 day under LD-30°C, but significant
increased after 5 day under LD-30°C. GO analysis of genes in cluster 3 displayed the major subcategory in “molecular function” was function on carbohydrate binding. The
major subcategories in “cellular component” were located at plasma membrane and cell
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wall. The major subcategories in “biological process” were related to defense response
and response to organic substance. Noteworthily, abundant genes in cluster 3 were
predicted to encode various members of stress-related transcription factors, including
AtGSTF3 (At2g02930) and PR5 (At1g75040). AtGSTF3 encoded glutathione
transferase. PR5 encoded thaumatin-like protein and involved in response to pathogens.
Furthermore, they all displayed drastic decreased expression levels after 1 day under
LD-30°C and increased after 5 day under LD-30°C.It suggests that they were response
to temperature stimulus and response to pathogens at the late phase.
Cluster 4:
In Fig. 21B and Table. 4, there were 63 transcripts in cluster 4, and their expression
levels increased highly in wild type compared with OgAPXOX after 5 day under
LD-30°C. GO analysis of genes in cluster 4 displayed the major subcategory in
“molecular function” was provided with transmembrane transporter activity. The major
subcategory in “cellular component” was located at integral membrane. The major
subcategories in “biological process” were related to temperature stimulus and response
to reactive oxygen species. Noteworthily, abundant genes in cluster 4 were predicted to
encode heat shock transcription factors, including, At5g37670, AtHSP70 (At3g12580),
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HSP70T-2, At2g19310, HSP81-.1 (At5g52640). Furthermore, they all displayed striking
increase expression levels in transgenic plants after 5 day under LD-30°C, and suggests
that they promote trigger the heat response by sensing AsA state.
Cluster 5:
In Fig. 21B and Table. 5, there were 92 transcripts in cluster 5, and displayed
overall tends of increased gene expression in control and transgenic plants under
LD-30°C compared with cluster 4. GO analysis of genes in cluster 5 displayed the major subcategory in “molecular function” was response to oxidative stress. The major
subcategory in “cellular component” was located at cell wall. The major subcategories
in “biological process” were related to organic substance and response to oxidation
reduction. Noteworthily, abundant genes in cluster 5 were predicted to detoxification
process, including, CYP96A4 (At5g52320), CYP79B2 (At4g39950), CYP81F4
(At4g37410), CYP83B1 (At4g31500), CYP71B3 (At3g26220), CYP89A9 (At3g03470)
and CSD2 (At2g28190). Furthermore, they all displayed increased expression levels
under LD-30°C and suggest oxidoreduction would be responsive to change of growth
temperature and AsA state.
Cluster 6:
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In Fig. 21B and Table. 6, total of 73 transcripts in cluster 6 displayed steady
expression pattern in control and transgenic plants after 1 day under LD-30°C, but
significant increased in transgenic plants after 5 day under LD-30°C. GO analysis of genes in cluster 6 displayed the major subcategory in “molecular function” was function
on metal ion binding. The major subcategory in cellular component was located at
external encapsulating structure. The major subcategories in “biological process” were
related to oxidation reduction and response to organic substance. Noteworthily,
At3g47480 was predicted to encode calcium-binding protein. Interestingly, At3g47480
displayed striking increase in OgAPXOX plants after 5 day under LD-30°C. It was
suggested that calcium-triggered signal cascade could sense the redox state.
Cluster 7:
In Fig. 21B and Table. 7, total of 49 transcripts in cluster 7 displayed slightly
decreased expression pattern after 1 day under LD-30°C, but significant increased after
5 day under LD-30°C. GO analysis of genes in cluster 7 displayed the major subcategories in “molecular function” were provided with transcription regulator
activity and transcription factor activity. The major subcategory in “cellular component”
was located at intrinsic membrane. The major subcategories in “biological process”
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were related to endogenous stimulus, defense response, response to organic substance
and response to carbohydrate stimulus. Noteworthily, abundant genes in cluster 7 were
predicted to encode various members of stress response transcription factors, including
AtERF6 (At4g17490), ATERF-2(At5g47220), AtACS11 (At4g08040) and AtACS6
(At4g11280). All of them played important role in ACC synthesis. Interestingly,
transgenic Arabidopsis overexpressing AtMYB44 was more sensitive to ABA and had a
more rapid ABA-induced stomatal closure response than wild type and atmyb44
knockout plants. AtMYB44 showed slightly decreased after 1 day under LD-30°C, but,
induced drastically after 5 day under LD-30°C. It suggests that the stress
phytohormones, including of ethylene and ABA, would be retrieved in response to AsA
status under elevated growth temperature.
Cluster 8:
In Fig. 21B and Table. 8, cluster 8 contains 55 transcripts that displayed more
significantly decreased expression pattern after 1 day under LD-30°C, but significantly
increased after 5 day under LD-30°C compared with cluster 7. GO of genes in cluster 8 displayed the major subcategories in “molecular function” was function on ATP binding.
The major subcategories in “cellular component” were located at plasma membrane,
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cell wall. The major subcategories in “biological process” were related to defense
response and response to organic substance. Noteworthily, abundant genes in cluster 8
were predicted to encode various members of pathogenesis-related transcription factors,
including AtPR1 (At2g14610), AtPR4 (At3g04720), PDF1.2 (At5g44420). Furthermore,
they all displayed drastic decreased expression levels after 1 day under LD-30°C, and
increased after 5 day under LD-30°C, and suggested that they were response to
thermal-induced acclimation and sensing the redox state at the late phase to reduce
injury by pathogen presumably.
Cluster 9:
In Fig. 21B and Table. 9, total of 62 transcripts in cluster 9 displayed steady
expression pattern after 1 day under LD-30°C, but significantly increased in wild type
after 5 day under LD-30°C. GO of genes in cluster 9 displayed the major subcategory in
“molecular function” was function on cation binding. The major subcategory in
“cellular component” was located at vacuole. The major subcategories in “biological
process” were related to defense response, response to hormone stimulus and response
to carbohydrate stimulus. Noteworthily, abundant genes among cluster 9 were predicted
to encode various members of MYB transcription factors, including AtMYB73
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(At4g37260), MYB77 (At3g50060). Interestingly, MYB77 modulated auxin signal
transduction (Shin et al., 2007). AtMYB73 was response to salicylic acid stimulus.
MYB77 was showed slightly decreased after 1 day under LD-30°C, but, induced
drastically after 5 day under LD-30°C. It suggests that the auxin and salicylic acid
stimulus in plant process was trigger by downstream genes under elevated growth
temperature.
Cluster 10:
In Fig. 21B and Table. 10, cluster 10 contains 53 transcripts that displayed
significantly decreased expression pattern after 1 day under LD-30°C, but, significant
increased after 5 day under LD-30°C. GO of genes in cluster 10 displayed the major subcategories in “molecular function” was provided with serine/threonine kinase
activity. The major subcategory in “cellular component” was located in cytosol. The major subcategories in “biological process” were elated to hormone stimulus and
response to abiotic stimulus. Noteworthily, abundant genes in cluster 10 were predicted
to encode putative protein kinase, including CIPK20, AtCPK6 (At2g17290).
Furthermore, they all displayed drastically decreased expression levels after 1 day under
LD-30°C, and increased after 5 day under LD-30°C, and suggested response to
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thermal-induced acclimation.