<p>Phenylalanine
(Phe) is a proteinogenic aromatic amino acid that also serves as a precursor
for numerous primary and secondary metabolites in plants. Phe is synthesized
from chorismate, the final product of the shikimate pathway. In plants, Phe is predominantly
synthesized in the plastids via the arogenate pathway, while most Phe-derived
compounds are produced in the cytoplasm, requiring exportation of Phe from
plastids to the cytosol. Here, we provided genetic evidences that a<i> Petunia hybrida</i> plastidial cationic
amino acid transporter (PhpCAT) participates in the exportation of Phe from
plastids, as well as regulation of carbon flux through Phe biosynthesis.</p>
<p> By using reverse genetics, we demonstrated
that a petunia phenylpyruvate aminotransferase (PhPPY-AT) is able to convert
phenylpyruvate to Phe in the cytosol <i>in
vivo</i>, and that a cytosolic chorismate mutase (CM2), which converts
chorismate to prephenate, directs carbon flux from the plastidial Phe
biosynthesis pathway towards the cytosolic pathway. Downregulation of <i>PhPPY-AT</i> and <i>PhCM2</i> resulted in significant decreases in Phe levels and emission
of Phe-derived volatiles in petunia flowers, respectively. Metabolic flux
analysis showed that the carbon flux through the cytosolic Phe biosynthesis
pathway is significantly lower in <i>PhCM2</i>
RNAi petunia flowers relative to wild type control. We also demonstrated that
the conversion of prephenate to phenylpyruvate in the cytosol is catalyzed by a
cytosolic prephenate dehydratase (PDT) produced from an alternative
transcription start site of a known plastidial arogenate dehydratase (ADT). These
results suggest that a microbial-like phenylpyruvate pathway for Phe
biosynthesis operates in the cytosol of plant cells and the cytosolic pathway
splits from the plastidial pathway at chorismate.</p>
<p> To evaluate the metabolic potential of
the cytosolic phenylpyruvate pathway, <i>PhCM2
</i>overexpressing transgenic petunia plants were generated. Unexpectedly, Phe
levels and emission of Phe-derived volatiles were both reduced, even though the
flux through the cytosolic pathway was increased relative to wild type control.
Electron microscopy, metabolic profiling and metabolic flux analysis revealed
that the number of leucoplasts, starch levels and flux through the plastidial
pathway were all reduced in <i>PhCM2</i>
overexpression lines, while the concentrations of auxin and its biosynthetic
intermediate, indole-3-pyruvic acid (IPA), were elevated. Overexpression of
Arabidopsis aminotransferase VAS1, which converts IPA to Trp, in <i>PhCM2</i> overexpression petunia background
recovered Phe levels and Phe-derived volatiles emission. These results indicate
that there exists a metabolic crosstalk between cytosolic Phe production and
Trp-dependent auxin biosynthesis .</p>
<p> Our research completed the
post-chorismate cytosolic Phe biosynthesis pathway in plants and revealed
possible metabolic crosstalk between cytosolic Phe production and auxin
biosynthesis in plant cells, providing targets for future genetic modification
of metabolites in plants.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/8020661 |
| Date | 15 May 2019 |
| Creators | Yichun Qian (5930168) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Discovery_of_Cytosolic_Phenylalanine_Biosynthetic_Pathway_in_Plants/8020661 |
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