Part:BBa_K4452005
prSSG1 transit peptide
This transit peptide was identified for implementing root magnetotropism by overexpressing ferritin in statoliths of columella cells in Arabidopsis thaliana. While no statolith import sequences have been validated, this transit peptide was selected as a candidate sequence for importing ferritin into statoliths.
Background
To restore directional root growth in microgravity, Hopkins iGEM 2022 proposed that the existing gravitropic mechanisms can be engineered to respond to an artificial cue. We set out to engineer roots to grow in the direction of magnetic field gradients: magnetotropism.
Plants sense gravity via statoliths—starch-laden organelles in root tip columella cells—which sediment due to their weight. Statolith sedimentation triggers changes in the efflux of auxin, a universal plant hormone that induces plant cell elongation. Polarized auxin accumulation along the upper and lower sides of roots causes differential elongation of cells, guiding root growth in the direction of gravity.
We predicted that filling statoliths with iron-loading proteins, like ferritin, would allow the statoliths to move in response to a magnetic gradient. For our project we designed a genetic construct that allows for ferritin to be overexpressed in Arabidopsis and imported into statoliths.
Biology
In addition to overexpressing ferritin in the correct cells, we need to import the ferritin into the correct intracellular compartments, i.e the statoliths.
Statoliths are starch-laden organelles in plant cells. The class of organelles in plants that are involved in food synthesis and storage are called plastids. The plastids that are responsible for food synthesis usually contain pigments. Chloroplasts are an example of plastids that are involved in food synthesis and they contain the green pigment chlorophyll.
Plastids without pigments are called leucoplasts. Leucoplasts are responsible for food storage and are renamed for the type of food they store. Leucoplasts that store starch are called amyloplasts, and thus, statoliths are a type of amyloplast.
Proteins that originate from nuclear transcripts must have a transit peptide (TP) sequence on their N terminus, which will bind to translocons on the plastid membrane after its initial translation. The rest of the protein will then be translated directly into the plastid. No validated statolith import sequences are known, so we selected sequences to test. We searched for papers that mentioned root amyloplast TPs, and searched the Gene Ontology (GO) Resource for proteins known to be in the statolith that had annotated TPs.
Candidate: prSSG1 Transit Peptide
Granule-bound starch synthase 1 (SSG1) is an enzyme involved in the starch biosynthesis pathway, specifically in the synthesis of amylose, one of the two distinct polymers within statolith starch granules. SSG1 is expressed in roots, but is more highly expressed in leaves, where it exhibits circadian up-regulation during the day [1, 2, 3]. The transit sequence of prSSG1 is annotated on https://www.uniprot.org/uniprotkb/Q9MAQ0/feature-viewer. Upon advice from Dr. Hsou-min Li, the principal investigator of the Chu et al. [4], we used the first 60 residues of the preproteins pulled from GO instead of the sequence that was annotated, as Dr. Li reported that TPs are not typically shorter than 50 residues.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] Seung D, Soyk S, Coiro M, Maier BA, Eicke S, et al. (2015) PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis. PLOS Biology 13(2): e1002080. https://doi.org/10.1371/journal.pbio.1002080
[2] Tenorio, G., Orea, A., Romero, J.M. et al. Oscillation of mRNA level and activity of granule-bound starch synthase I in Arabidopsis leaves during the day/night cycle. Plant Mol Biol 51, 949–958 (2003). https://doi.org/10.1023/A:1023053420632
[3] Steven M. Smith, Daniel C. Fulton, et al. Diurnal Changes in the Transcriptome Encoding Enzymes of Starch Metabolism Provide Evidence for Both Transcriptional and Posttranscriptional Regulation of Starch Metabolism in Arabidopsis Leaves, Plant Physiology, Volume 136, Issue 1, September 2004, Pages 2687–2699, https://doi.org/10.1104/pp.104.044347
[4] Chiung-Chih Chu, Krishna Swamy, Hsou-min Li, Tissue-Specific Regulation of Plastid Protein Import via Transit-Peptide Motifs, The Plant Cell, Volume 32, Issue 4, April 2020, Pages 1204–1217, https://doi.org/10.1105/tpc.19.00702
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