In JoVE (1)
Other Publications (7)
- Journal of Experimental Botany
- Molecular Genetics and Genomics : MGG
- The Journal of Biological Chemistry
- Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
- The Plant Journal : for Cell and Molecular Biology
- Plant Biotechnology Journal
- Plant Physiology and Biochemistry : PPB / SociÃ©tÃ© FranÃ§aise De Physiologie VÃ©gÃ©tale
Articles by Susanne E. Kohalmi in JoVE
Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System Gang Tian1, Qing Lu2, Li Zhang2, Susanne E. Kohalmi1, Yuhai Cui2 1Department of Biology, University of Western Ontario, 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.
Other articles by Susanne E. Kohalmi on PubMed
Mutation in the Ap2-6 Allele Causes Recognition of a Cryptic Splice Site Journal of Experimental Botany. Dec, 2003 | Pubmed ID: 14563835 Mutations in the homeotic gene APETALA2 of Arabidopsis thaliana cause severe developmental alterations, most prominently homeotic floral organ replacements from petals to carpels and petals to stamens in the outer two floral whorls. To date, ten different alleles have been identified conferring phenotypes of various degrees. Of these ten alleles, only three have been characterized at the sequence level. The identification of the sequence alteration in the ap2-6 allele is reported here. In ap2-6 a single G.C to A.T transition occurred at the 3' end of intron 6 (position 1342) which leads to a dinucleotide loss at the mRNA level. This change is consistent with the G.C to A.T transition destroying a conserved dinucleotide motif (AG) required for proper splice recognition and with the resulting recognition of the next available downstream AG dinucleotide which in AP2 is immediately adjacent to the authentic 3' splice site. The dinucleotide loss will cause a frameshift, the translation of three incorrect amino acids and a premature stop codon resulting in a truncation of the AP2 sequence within the AP2-R2 domain. Such a truncation is predicted to impact severely on the function of AP2 and is consistent with the observed phenotype.
Cytochrome F from the Antarctic Psychrophile, Chlamydomonas Raudensis UWO 241: Structure, Sequence, and Complementation in the Mesophile, Chlamydomonas Reinhardtii Molecular Genetics and Genomics : MGG. Apr, 2006 | Pubmed ID: 16425016 Although cytochrome f from the Antarctic psychrophile, Chlamydomonas raudensis UWO 241, exhibits a lower apparent molecular mass (34 kD) than that of the mesophile C. reinhardtii (41 kD) based on SDS-PAGE, both proteins are comparable in calculated molecular mass and show 79% identity in amino acid sequence. The difference in apparent molecular mass was maintained after expression of petA from both Chlamydomonas species in either E. coli or a C. reinhardtii DeltapetA mutant and after substitution of a unique third cysteine-292 to phenylalanine in the psychrophilic cytochrome f. Moreover, the heme of the psychrophilic form of cytochrome f was less stable upon heating than that of the mesophile. In contrast to C. raudensis, a C. reinhardtii DeltapetA mutant transformed with petA from C. raudensis exhibited the ability to undergo state transitions and a capacity for intersystem electron transport comparable to that of C. reinhardtii wild type. However, the C. reinhardtii petA transformants accumulated lower levels of cytochrome b ( 6 ) /f complexes and exhibited lower light saturated rates of O(2) evolution than C. reinhardtii wild type. We show that the presence of an altered form of cytochrome f in C. raudensis does not account for its inability to undergo state transitions or its impaired capacity for intersystem electron transport as previously suggested. A combined survey of the apparent molecular mass, thermal stability and amino acid sequences of cytochrome f from a broad range of mesophilic species shows unequivocally that the observed differences in cytochrome f structure are not related to psychrophilly. Thus, caution must be exercised in relating differences in amino acid sequence and thermal stability to adaptation to cold environments.
Phenylalanine Biosynthesis in Arabidopsis Thaliana. Identification and Characterization of Arogenate Dehydratases The Journal of Biological Chemistry. Oct, 2007 | Pubmed ID: 17726025 There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.
The Small Domain of Cytochrome F from the Psychrophile Chlamydomonas Raudensis UWO 241 Modulates the Apparent Molecular Mass and Decreases the Accumulation of Cytochrome F in the Mesophile Chlamydomonas Reinhardtii Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire. Oct, 2007 | Pubmed ID: 17901903 Cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 has a lower thermostability of its c-type heme and an apparent molecular mass that is 7 kDa lower than that of the model mesophilic green alga Chlamydomonas reinhardtii. We combined chloroplast transformation, site-directed mutagensis, and the creation of chimeric fusion constructs to assess the contribution of specific domains and (or) amino acids residues to the structure, stability, and accumulation of cytochrome f, as well as its function in photosynthetic intersystem electron transport. We demonstrate that differences in the amino acid sequence of the small domain and specific charged amino acids in the large domain of cytochrome f alter the physical properties of this protein but do not affect either the thermostability of the c-type heme, the apparent half-life of cytochrome f in the presence of the chloroplastic protein synthesis inhibitor chloramphenicol, or the capacity for photosynthetic intersystem electron transport, measured as e-/P700. However, pulse-labeling with [14C]acetate, combined with immunoblotting, indicated that the negative autoregulation of cytochrome f accumulation observed in mesophilic C. reinhardtii transformed with chimeric constructs from the psychrophile was likely the result of the defective association of the chimeric forms of cytochrome f with the other subunits of the cytochrome b6/f complex native to the C. reinhardtii wild type. These results are discussed in terms of the unique fatty acid composition of the thylakoid membranes of C. raudensis UWO 241 adapted to cold environments.
Arabidopsis Homolog of the Yeast TREX-2 MRNA Export Complex: Components and Anchoring Nucleoporin The Plant Journal : for Cell and Molecular Biology. Jan, 2010 | Pubmed ID: 19843313 Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.
Plant-derived Recombinant Human Serum Transferrin Demonstrates Multiple Functions Plant Biotechnology Journal. May, 2010 | Pubmed ID: 20432512 Human serum transferrin (hTf) is the major iron-binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high-quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 microg/g fresh leaf weight). Furthermore, plant-derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum-free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell-specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes.To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon-like peptide 1 (GLP-1) or its derivative in plants. Here, we show that plant-derived hTf-GLP-1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro.
Complementation of the Pha2 Yeast Mutant Suggests Functional Differences for Arogenate Dehydratases from Arabidopsis Thaliana Plant Physiology and Biochemistry : PPB / SociÃ©tÃ© FranÃ§aise De Physiologie VÃ©gÃ©tale. Aug, 2011 | Pubmed ID: 21388819 The final steps of phenylalanine (Phe) biosynthesis in bacteria, fungi and plants can occur via phenylpyruvate or arogenate intermediates. These routes are determined by the presence of prephenate dehydratase (PDT, EC18.104.22.168), which forms phenylpyruvate from prephenate, or arogenate dehydratase (ADT, EC22.214.171.124), which forms phenylalanine directly from arogenate. We compared sequences from select yeast species to those of Arabidopsis thaliana. The in silico analysis showed that plant ADTs and yeast PDTs share many common features allowing them to act as dehydratase/decarboxylases. However, plant and yeast sequences clearly group independently conferring distinct substrate specificities. Complementation of the Saccharomyces cerevisiae pha2 mutant, which lacks PDT activity and cannot grow in the absence of exogenous Phe, was used to test the PDT activity of A. thaliana ADTs in vivo. Previous biochemical characterization showed that all six AtADTs had high catalytic activity with arogenate as a substrate, while AtADT1, AtADT2 and AtADT6 also had limited activity with prephenate. Consistent with these results, the complementation test showed AtADT2 readily recovered the pha2 phenotype after âˆ¼6 days growth at 30 Â°C, while AtADT1 required âˆ¼13 days to show visible growth. By contrast, AtADT6 (lowest PDT activity) and AtADT3-5 (no PDT activity) were unable to recover the phenotype. These results suggest that only AtADT1 and AtADT2, but not the other four ADTs from Arabidopsis, have functional PDT activity in vivo, showing that there are two functional distinct groups. We hypothesize that plant ADTs have evolved to use the arogenate route for Phe synthesis while keeping some residual PDT activity.