6 Supplementary Results
6.5 Analysis of Stably Expressing GRX480 lines in planta
6.5.3 Effect of GRX480 over-expression on Arabidopsis defense genes
The accumulation of the anti-fungal protein PDF1.2, which is under normal circumstances induced by jasmonic acid, was compromised in the presence of constitutively high levels of GRX480 (Figure 4A, manuscript) which is also inducible by SA (Figure 1B of manuscript). In order to find out the array of possible transcription factors whose expression may be regulated by Atg28480 and possibly influence the expression of PDF1.2 and other target genes, a macro-array experiment was carried out on a REGIA array, on which different transcription factors which regulate diverse stress responses were spotted.
The RNA used as a probe was taken from an experiment where wild-type control plants and plants stably expressing the GRX480 gene under the influence of the 35SCaMV promoter were treated under the same conditions with 20µM meJA. RNA from the sample time point 4 hours post induction was used for the array experiment (see manuscript, Figure 4A).
The candidate genes down regulated by GRX480 over-expression among the confirmed plant defensin protein PDF1.2a, included a Transcription factor II homolog, a bHLH protein, and a bZIP protein BZ02H2. Though these were not as significantly reduced on the stress array as PDF1.2, their relevance still has to be investigated by northern blotting.
The candidate genes upregulated by GRX480 over-expression included a salt-tolerance zinc finger protein, a CONSTANS B-box zinc finger family protein, a jasmonic acid regulatory protein, a disease resistance protein EDS1, a flavanone 3-hydroxylase (FH3), PAD4, a NAM-like Protein (At4g27410), two WRKY family transcription factors, a heat shock transcription factor HSF4, zinc finger protein Zat12 and a MYB96TF-like protein.
(See Table 6.5A).
Their relevance also has to be further investigated by northern blotting.
The GENVESTIGATOR toolkit (Zimmermann et al., 2004) was nevertheless used to compare the expression pattern of At1g2840 and the genes differentially regulated as observed in the array. Expression patterns during different conditions of biotic/abiotic
stress as well as tissue specific expression patterns from results of different microarray experiments were used for comparison. The results are illustrated on Tables 6.5B-E.
Table 6.5A. Genes which are differentially regulated by over expression of GRX480 in REGIA macro array of stress related transcription factors.
Genes Downregulated by overexpressing GRX480 AGI Code Annotation/Description
At4g31720 Transcription initiation factor IID protein At5g24800 bZIP transcription factor family protein At5g44420 Plant defensin protein, PDF1.2a,
Encodes an ethylene- and jasmonate-responsive plant defensin. mRNA levels are not responsive to salicylic acid treatment.
At5g65640 Basic helix-loop-helix (bHLH) family protein Genes Upregulated by overexpressing GRX480
AGI Code Annotation/Description
At1g27730 Salt tolerance zinc finger (C2H2-type) protein (ZAT10) responsive to chitin oligomers.
At1g62300 Wrky6 transcription factor
At1g78600 Zinc finger (B-box type) family protein At3g15500 Jasmonic acid regulatory protein At3g48090 Disease resistance protein EDS1
Component of R gene-mediated disease resistance in Arabidopsis thaliana with homology to eukaryotic lipases.
At3g51240 Flavanone 3-hydroxylase (FH3)/ Naringenin 3-dioxygenase
Encodes flavanone 3-hydroxylase that is coordinately expressed with chalcone synthase and chalcone isomerases. Regulates flavonoid biosynthesis.
At3g52430 Phytoalexin-deficient 4 protein (PAD4),
Encodes a lipase-like gene that is important for salicylic acid signaling and function in resistance (R) gene-mediated and basal plant disease resistance. PAD4 can interact directly with EDS1, another disease resistance signaling protein. Expressed at elevated level in response to green peach aphid (GPA) feeding, and modulates the GPA feeding-induced leaf senescence through a mechanism that doesn't require camalexin synthesis and salicylic acid (SA) signaling.
At4g27410 No apical meristem (NAM) family protein (RD26) At4g31800 WRKY family transcription factor (WRKY18) At4g36990 Heat shock transcription factor HSF4
Encodes a protein whose sequence is similar to heat shock factors that regulate the expression of heat shock proteins. Transcript level is increased in response to heat shock. However, over-expression of this gene did not result in the increase or decrease of heat shock proteins.
At5g59820 Zinc finger (C2H2 type) family protein (ZAT12)
Encodes a zinc finger protein involved in high light and cold acclimation At5g62470 myb family transcription factor (MYB96)
Table 6.5B Differential expression of genes down-regulated by the stable expression of GRX480, after 20uM JA induction: How they are normally regulated under biotic and abiotic stress induced conditions.
Treatment
AT4G31720 AT5G65640 AT5G24800 AT5G44420 AT1G28480
Table shows the regulation of different genes (In rows with their AGI codes indicated on the last column to the right) by different stress situations (columns, with the biotic and abiotic stress types indicated on the first row on top).
See color key below.
Table 6.5C. Differential expression of genes down-regulated by the stable expression of GRX480, after 20uM JA induction: How they are normally expressed in different tissues.
AGI/Links/Annotation AT4G31720 (TFIID)
AT5G24800 bZIP transcription factor AT5G65640 (bHLH)
AT1G28480 ]glutaredoxin AT5G44420 (PDF1.2a)
Table shows the regulation of different genes (In rows with their AGI codes indicated on the last column to the right) in different plant tissue types (columns, with tissue type indicated on the first row on top).
See Color Key below.
Table 6.5D. Differential expression of genes up-regulated by the stable expression of GRX480, after 20uM JA induction: How they are normally regulated under biotic and abiotic stress inducible conditions.
Treatment
At1g27730 AT1G28480 AT5G59820 AT1G78600 AT5G62470 AT3G51240 AT3G48090 AT4G27410 AT4G31800 AT3G52430 AT3G15500 AT4G36990 AT1G62300
Table shows the regulation of different genes (In rows with their AGI codes indicated on the last column to the right) by different stress situations (columns, with the biotic and abiotic stress types indicated on the first row on top).
See color key below.
Table 6.5E. Differential expression of genes up-regulated by the stable expression of GRX480, after 20uM JA induction: How they are normally expressed in different tissues.
AT3G15500 (NAC3) AT4G27410, (RD26) AT5G59820, (ZAT12) AT1G27730, (ZAT10)
AT4G31800, WRKY family transcription factor AT1G28480, glutaredoxin family protein AT1G62300, WRKY family transcription factor AT4G36990, (HSF4)
AT3G48090, (EDS1) AT3G52430, (PAD4)
AT1G78600, zinc finger (B-box type) family protein AT3G51240, (F3H)/ N3D
AT5G62470, (MYB96)
Table shows the regulation of different genes (In rows with their AGI codes indicated on the last column to the right) in different plant tissue types (columns, with tissue type indicated on the first row on top).
See Color Key below.
Table Legends for TABLES 6.5B-E
>=95%
>=90% < 95%
>=85% < 90%
>=80% < 85%
>=75% < 80%
>=70% < 75%
>=65% < 70%
>=60% < 65%
>=55% < 60%
>=50% < 55%
>=45% < 50%
>=40% < 45%
>=35% < 40%
>=30% < 35%
>=25% < 30%
>=20% < 25%
>=15% < 20%
>=10% < 15%
>=5% < 10%
>=0% < 5%
Color Keys: For the red-green scheme, red indicates that "the signal intensity of the treatment is higher than signal intensity of the corresponding control", and green means that "the signal intensity of the treatment is lower than signal intensity of the corresponding control".
For the blue-white scheme, all gene-level profiles were normalized for coloring such that for each gene the highest signal intensity obtains value 100% (dark blue) and absence of signal obtains value 0 % (white).
6.5.4 Generation of lines stably over-expressing mutant derivatives of GRX480