Differential expression of glutaredoxins

The expression pattern of GRX480 and related glutaredoxins was analyzed based on information available from the Arabidopsis microarray database. The GENVESTIGATOR toolkit was used to compare the expression patterns under different conditions of biotic/abiotic stress, during different stages of development as well as expression patterns occurring in specific tissues (Zimmermann et al., 2004; https://

www.genevestigator.ethz.ch/at/index.php?page=home).

6.2.1 Stress induced differential expression of GRX480 and related glutaredoxins The AGI codes for genes producing significant alignment to At1g28480 were input into the Meta-Analyzer program. The following were not available in the GeneExpress database: At4g15670, At2g47870, At4g28730, At5g01420 and At5g06470. The genes coding for proteins whose active site residues were not conserved (see Figure 6.1B) were also left out in the analysis.

It is interesting to note that At1g03850 which is most closely related to GRX480 is also inducible by pathogens, cyclohexamide and SA, in a similar pattern as GRX480. GRX480 is more differentially induced by salt stress in the roots.

At4g15690 is not inducible by pathogen P. syringae, but is SA inducible. GRX480 has 1.8 and 1.5 fold stronger inducibility by SA than At1g03850 and At4g15690 respectively.

Pathogen induction by GRX480 is also 1.3 fold stronger than by At1g03850.

Even though these array data show a similar induction of GRX480 by SA and JA, northern blot data show weak induction in the presence of JA, stronger induction in the presence of SA (Figure 6.2), and synergistically stronger induction in the presence of both SA and JA (see Figure 5 of manuscript). The induction by SA is nevertheless 5-11 fold weaker than that by cyclohexamide ( also reported in northern analysis by Thurow, unpublished) in wild type Columbia plants. Both SA and JA pathways seem to be important in inducing the accumulation of GRX480 transcript, especially as induction is also dependent on the COI protein (Thurow, unpublished).

It is also worth noting that GRX480 is uniquely regulated by cyclohexamide, unlike At1g03850 and At4g15690 which are between 40 and 60 times less inducible than GRX480 after cyclohexamide treatment.

Table 6.2A Differential expression of GRX480 and related glutaredoxins under biotic and abiotic stress induced conditions.

Treatment

AGI CODE AT2G30540 AT4G33040 AT3G62960 AT5G14070 AT1G06830 AT2G38270 AT2G20270 AT3G54900 AT3G15660 AT5G20500 AT4G04950 AT3G02260 AT5G40370 AT5G11930 AT5G03870 AT3G62930 AT5G63030 AT3G62930 AT5G63030 AT1G77370 AT4G15700 AT4G15660 AT5G18600 AT4G15690 AT2G47880 AT1G32760 AT4G15680 AT1G03020 AT1G64500 AT3G02000 AT3G62950 AT1G03850 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).

Key(Below): 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".

0 4 8 12 24 0 4 8 12 24 0 4 8 12 24 0 4 8 12 24 Time SA JA 2,4D ETOH

GRX

RNA

Figure 6.2: Expression analysis of GRX480

Three weeks old A. thaliana Col-O plants were treated with either 1mM SA, 20µM meJA, 0.1mM 2,D or 0.01% Ethanol control for the time periods indicated above the lanes. 20 µg of RNA was loaded and the blot was hybridized with probes for GRX480 (GRX). Ethidium bromide stained RNA is shown to document equal loading.

6.2.2 Developmental stage specific expression of GRX480 and related glutaredoxins

The AGI codes for genes producing significant alignment to At1g28480 were input into the Meta-Analyzer program except for At4g15670, At2g47870, At4g28730, At5g01420, At5g06470 and the genes coding for proteins whose active site residues were not conserved.

The developmental stage specific expression pattern of the input genes was displayed (see Table 6.2B). GRX480 basal expression peaks during Arabidopsis development occur during stages 1.03-1.05 (16-18 days old) when there are 3-5 rosette leaves; and stage 3.7 (26-28 days old) when the rosette leaves are about 70% of their final size. The amounts accumulating in the later are higher and might explain the reason why as-1::GUS plants which are 4 weeks old and above are unresponsive to auxin induced expression of the GUS transcript (results not shown), since high amounts of GRX480 repress as-1 mediated expression in as-1::GUS plants. This is nevertheless only speculative.

Table 6.2B. Differential expression of GRX480 and related glutaredoxins during development.

Table shows the regulation of different genes (In rows with their AGI codes indicated on the last column to the right) during different developmental stages (columns, with age in days indicated on the first row on top). GRX480 is indicated in red.

Color Key(Right): 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).

Even though the glutaredoxins are regulated in expression during diverse developmental stages, a few cluster during specific developmental stages. At3g02000 (ROXY1), known for regulating flower development clusters along with others around days 21-25 of development.

6.2.3 Tissue specific expression of GRX480 and related glutaredoxins

AGI codes for genes producing significant alignment to At1g28480 were input into the Meta-Analyzer as in section 6.2.1 above. The expression of GRX480 as well as other glutaredoxins in different tissue types is indicated on Table 6.2C below.

Table 6.2C. Differential expression of GRX480 and related glutaredoxins in different plant tissues.

AGI CODE AT3G15660 AT4G04950 AT1G32760 AT3G02260 AT1G77370 AT5G40370 AT5G20500 AT2G20270 AT3G54900 AT4G33040 AT5G63030 AT2G47880 AT1G64500 AT3G62960 AT5G18600 AT4G15690 AT4G15680 AT4G15700 AT1G06830 AT3G62950 AT1G03020 AT4G15660 AT3G62930 AT5G14070 AT2G30540 AT2G38270 AT3G02000 AT5G03870 AT5G11930 AT1G03850 AT1G28480

The table above 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). GRX480 is indicated in red.

Key: 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). (See Key of Table 6.2B above).

The highest expression levels of GRX480 are found in the sepals and in the ridicule.

These are stronger, compared to expression levels in the roots and adult leaves. This is somewhat different from the expression pattern of a classic glutaredoxin like At5g20455 which is widely and highly expressed in most tissues.