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The atToc159 receptor complex represents a critical point in the biosynthesis of jasmonic acid
Author and co-authors: Danielle Sprout, Kayla Duffield, Samantha Workman
Faculty Advisor: Dr. Afitlhile
Department of Biological Sciences
Objective
We evaluated the ability of ppi2 mutant to accumulate
mRNA that encode enzymes that function in the JA
pathway. We also measured transcript levels of the JA
responsive gene, PDF 1.2 and JAZ1, which encodes a
protein that represses JA responsive genes.
Hypothesis
The atToc159 receptor complex is a major route in the
import of LOX-2, AOS and AOC, which are enzymes that
function in the initial stages of the JA pathway. We expect
the wounded mutant to accumulate low levels of mRNA
that encode LOX-2, AOS, OPR-3, and the JA responsive
gene, PDF1.2.
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Wt, uw
Wt, w
Mt, uw
Mt, w
5
Relative fold change
4
3
2
1
0
LOX-2
heterozygous
ppi2
AOS
OPR-3
Figure 1. Expression of LOX-2, AOS and OPR3 genes in the unwounded and wounded leaves of ppi2
mutant and wild type plants. Data represent the mean ± SEM of 4 replicates.
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Discussion
In the wounded wild type, the expression of LOX-2, AOS, OPR-3,
and the JA responsive gene PDF 1.2 was upregulated. Expression
of JAZ1 was comparable between the unwounded and wounded
tissue, which suggests that the synthesis and degradation of JAZ1
protein was tightly regulated. However, in the wounded ppi2
mutant the expression of LOX-2, AOS, and OPR-3 was suppressed.
Interestingly, in ppi2 transcripts for the jasmonate responsive
gene, PDF1.2 did not accumulate above the control level. This
observation suggests that in the wounded mutant, JA or JA-Ile did
not accumulate to levels high enough to induce the expression of
PDF1.2 gene. The data indicate that in the absence of Toc159
receptor, enzymes that function in the initial stages of the JA
pathway were not efficiently imported into the chloroplasts. We
conclude that atToc159 receptor is required in the import of
enzymes that function in the JA pathway.
Acknowledgments
Relative fold change
Introduction
When plants are eaten by insect herbivores or wounded
mechanically, the fatty acid linolenic acid (18:3) is
metabolized to produce the plant hormone, jasmonic
acid (JA), which accumulates to high levels in wounded
tissues. The JA pathway is initiated in the chloroplasts
and completed in the peroxisomes. JA is then exported
to the cytoplasm where it is conjugated to isoleucine to
form JA-Ile. The latter binds to its receptor, which induce
signals that turn on an array of plant defense genes,
including plant defensin, PDF1.2. Enzymes that function
in the JA pathway are encoded by genes that are
localized within the nucleus. Lipoxygenase-2 (LOX-2),
allene oxide synthase (AOS), and allene oxide cyclase
(AOC) are synthesized in the cytoplasm and imported
into the chloroplasts, while OPR-3 is imported into the
peroxisomes. Proteins that are synthesized in the
cytoplasm are recognized by receptors in membranes of
the chloroplasts and peroxisomes. Receptors on the
outer membrane of the chloroplast include atToc159,
atToc132, and atToc120. These receptors are found in
the same complex with Toc33/34 and Toc75; the latter is
a protein channel. Mutation in atToc159 receptor yields
plants with an albino phenotype, and are called plastid
protein import deficiency or ppi2.
Wt, uw
Wt, w
Mt, uw
4
3
2
1
0
PDF1.2
JAZ1
Figure 2. Expression of PDF1.2 and JAZ1 genes in the unwounded and wounded leaves of ppi2
mutant and wild type plants. Data represent the mean ± SEM of 4 replicates.
LOX-2
1
2
3
AOS
4
1
2
1
2
JAZ1
1
2
OPR-3
3
4
3
4
1
2
3
4
UCE
3
4
Dr. Sue Musser, for assistance in the analysis of the qPCR
data (WIU).
Dr. Matthew Smith for the gift of the ppi2 seeds (Canada).
Figure 3. RT-PCR analysis of the expression of LOX-2, AOS, OPR-3 and JAZ-1. Ubiquitin-conjugating
enzyme (UCE) is the internal control. 1: unwounded, wild type. 2: wounded, wild type. 3: unwounded,
ppi2. 4: wounded, ppi2.