Analysis of GRX RNAi lines

The gene expression of the GRX480 was suppressed in plants using the anti-sense (RNAi) approach by transforming plants with the full length cDNA of GRX480 cloned into a pFGC vector in the sense and anti-sense orientation (Abdallat, 2004). Four of these lines were analyzed to determine whether the ability of these plants to suppress the JA induced gene PDF1.2 would be compromised either in the absence of SA or in the presence of SA induction. These plants showed incomplete but reduced amounts of the accumulation of GRX480 transcript after SA induction (Figure 6.6A), consistent with the fact that the RNA construct was functional in the plants.

GRX_OEx

GRXEnd 100

80 60 40 20

100 80 60 40 20

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20 Total GRX

50uM JA 1mM SA

GRX_OEx GRX_End

AS1GUS AS1GUS GRX3-OEx GxRNAi7 GxRNAi8 GxRNAi9

0 4 8 24 0 2 0 2 0 2 0 2 0 2 Time (Hrs PI)

RNA Loading control

0 4 8 24 0 2 0 2 0 2 0 2 0 2 Time (Hrs PI)

Figure 6.6A. Analysis of GRX480 RNAi line, by inability of SA to induce transcript accumulation. Three week old GRX480 RNAi lines #7, #8 and #9, control line (as-1::GUS) as well as over-expressing line #3 were treated for 2hrs with 1mM SA by spraying. Control as-1::GUS plants were also treated with 50µM JA by floating in a solution of phosphate buffer, and samples collected at time points 0, 4, 8 and 24hrs. 20µg each of prepared RNA was separated on a denaturing agarose gel. After northern blotting, the membrane was hybridized with a radioactively labeled probe for detecting the At1g28480 (GRX) transcript (above).

The RNA loading control is based on EtBr staining. The transcript was also quantified using the TINA software for the relative levels of expression per amount of RNA blotted, for the over-expressing (GRX_OEx), endogenous (GRX_End) or total amount of GRX accumulating (graphs below).

Since constitutive expression of GRX480 led to a repression at the PDF1.2 promoter, we assumed that the absence of the same in RNAi lines might super-induce the PDF1.2

promoter activity. Based on this hypothesis, the RNAi lines were induced with meJA, but unlike expected, they were unable to super-induce the induction of PDF1.2 relative to wild type plants (Figure 6.6B).

(I)

Figure 6.6B. The induction kinetics of JA-inducible genes in GRX480 RNAi lines.

Three week old GRX480 RNAi lines#6, #7, #8 and #9; wild type control line (as-1::GUS) as well GRX480 over-expressing line #3 were induced with 20µM meJA by floatation in buffer. Samples were collected over time points 0, 8 and 12hrs. Total cell RNA from the respective lines was prepared and 20ug loaded and separated on denaturing gels. After northern blotting, membranes were consecutively hybridized with radioactively labeled probes for detecting the transcripts. The loading controls are indicated for each case.

(I)Expression of PDF1.2 and WRKY6 in the respective lines.

(II)Expression of LOX2 and VSP2 in the respective lines.

Other JA response genes (LOX2 and VSP2) were also not affected in the RNAi lines. The WRKY6 gene, which contains an as-1 like element in its promoter was also not significantly affected.

In the presence of inducible amounts of SA, RNAi lines were still able to some extent to suppress the accumulation of PDF1.2 in the presence of SA (Figure 6.6C).

(I)

4hrs, PDF1.2

24Hrs PDF1.2

0 SA JA SA/JA ETOH 0 SA JA SA/JA ETOH 0 SA JA SA/JA ETOH 0 JA 0 JA

AS1:GUS GRXRNAi 8 GRXRNAi 9 GRXOEx1 GRXOEx3

RNA

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(II)

0 SA JA SA/JA ETOH 0 SA JA SA/JA ETOH 0 SA JA SA/JA ETOH 0 JA 0 JA

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4Hrs, JA 24Hrs, JA

Figure 6.6C. Analysis of GRX480 dependent cross talk in RNAi lines.

Three week old GRX480 RNAi lines #8 and #9; wild type control line (as-1::GUS) as well GRX480 over-expressing lines #1 and #3 were induced with either 1mM SA/Ethanol (SA), 20µM meJA (JA), SA/JA (SJ) or 0.02% Ethanol(EH) for 4 and 24 hours. Un-induced plants (0) were also collected. After pooling and collecting an average of 10 plants per time point, 20µg each of prepared RNA was separated on a denaturing agarose gel. After northern blotting, the membrane was hybridized with a radioactively labeled probe for detecting the PDF1.2 transcript (I - above). The RNA loading control is based on EtBr staining.

The signals were quantified using TINA software, and normalized based on the loading/blotting controls, for all the gels. The relative signal intensities are indicated for each time point in the graph below (II).

Since the RNAi lines did not completely knock out the GRX480 transcript, it can be assumed that the basal amounts of transcript induced after SA treatment in the RNAi lines could still repress the induction of PDF1.2 by JA. When the amounts of residual GRX transcript was quantified using TINA software, it could be observed that they still accumulate up to 5 fold increased levels after SA induction compared to untreated plants,

and to about 10fold increased levels compared to meJA treated plants at 4hours after induction (Figure 6.6A). It was nevertheless observed that the RNAi line #9 with seemingly the strongest RNAi effect was least able to suppress the induction of PDF1.2 by JA in the presence of SA after 4hrs (Figure 6.6C).

The RNAi line #9 was also able to induce PDF1.2 stronger that wild type and other RNAi lines, at comparable time points. Though this was observed 3 times, it is not statistically significant (Figure 6.6B). It was therefore necessary to analyze knockout lines, which are completely compromised in their ability to induce GRX480, in order to better understand their contribution to the cross-talk on the JA pathway.