Measuring the Plant's Gene Expression Response

The results of the plant response and the insect feeding experiments demonstrated a physiological link between JAR1 enzyme activity and the insect feeding response. Extending the experiments to coi1 mutants suggested that multiple genes drove the JA response pathway. Direct study of the expression and control of these genes was possible. The Staswick team wanted to understand some details of the JAR1 gene expression that led to these JA modification differences. A method they could use to document gene expression difference was a northern blot hybridization. The northern blot hybridization is a method of measuring the amount of a specific mRNA molecule in a sample. The specific mRNA is detected by binding it to a special membrane, then probing it with radioactive DNA copies of the specific gene. DNA from the gene binds to the mRNA that was encoded by the gene during the transcription process. Therefore, northern blots reveal if a specific gene was transcribed or expressed in a sampled tissue and generally if the expression was at a high level (lots of radioactive signal on the blot) or low level (little signal on the blot). There are other methods plant biologists can use to obtain a mRNA level that can be quantified with a number.

Figure 12. Wound-induced JAR1 mRNA in wild-type plants (WT) and jar1 mutants. In the bottom two rectangles ethidium bromide stained rRNA is shown as corresponding blot hybridizations as a total RNA loading control. At 1 hours after wounding the wild type and jar1-1 have very think bands of mRNA. These bands reduce in intensity at each of the other time points -- 3, 6, and 12 hours. The jar1-1 signals are thicker than those of the wild-type at the same point in time and the band intensity tapers off more slowly than the wild-type such that the jar1-1 still have some band present at 12 hours whereas the wild-type does not.

Quiz

Question

The UNL scientists wanted to see how the JAR1 gene expression differed, if any, between mechanically wounded wild-type plants and jar1 mutants. To test this, they collected mRNA from each of the three at certain time intervals after initial wounding, and they performed northern blot hybridizations using a DNA probe specific for the JAR1 mRNA. The results of their blots are shown in Figure 12 in the top two rectangles (the bigger/darker the spot, the more mRNA levels). Based on the results of the northern blot hybridizations, which of the following is correct?

A. The jar1 mutant allele is transcribed at about the same level and pattern as the wild type. So the mutation impacts gene expression after the transcription process.

The northern blot experiment showed that the transcription pattern of the jar1 mutant allele was essentially the same as the wild type. This demonstrates that the jar1 mutant has its impact at the post-transcriptional level.

B. The jar1 mutant allele is transcribed at about the same level as the wild-type but does not reach the maximum level of transcription at the same time after wounding as the wild-type allele.

Sorry, try again!

C. The jar1 mutant allele is not transcribed.

Sorry, try again!

Looks Good! The northern blot experiment showed that the transcription pattern of the jar1 mutant allele was essentially the same as the wild type. This demonstrates that the jar1 mutant has its impact at the post-transcriptional level.

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