Investigating the Production of Foreign Membrane Proteins in Tobacco Chloroplasts: Expression of an Algal Plastid Terminal Oxidase

Abstract

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.

Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes

“…1d) and hardly set seed. Similar pale and retarded growth phenotypes have also been observed when the plastoquinol/plastid terminal oxidase from Chlamydomonas reinhardtii was expressed in tobacco chloroplasts and have been linked to enhanced susceptibility to chronic photoinhibition (Ahmad et al 2012b).

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“…Growth retardation was also observed when transplastomic expression of maltose-binding protein (MBP) reached 37 % of TSP, possibly due to impaired export of maltose from the chloroplast (Ahmad et al 2012a). Expression of a thylakoid membrane protein, the plastid/plastoquinol terminal oxidase (PTOX) from Chlamydomonas reinhardtii , within tobacco chloroplasts rendered plants sensitive to high light (Ahmad et al 2012b). Overall the current data suggest that the toxic effects of foreign proteins accumulating within photosynthetically active plastids might pose a strong limitation for this technology.…”

Impaired Mitochondrial Transcription Termination Disrupts the Stromal Redox Poise in Chlamydomonas

“…The C. reinhardtii nuclear genome encodes two PTOX genes, and PTOX2 was shown to represent the major oxidase controlling the PQ redox poise in the dark (HouilleVernes et al, 2011). Although PTOX1 activity is comparably low in dark-acclimated C. reinhardtii cells (Houille-Vernes et al, 2011), residual chlororespiratory activity in a PTOX2 knockout mutant together with the phenotype caused by heterologous Cr-PTOX1 expression in tobacco (Ahmad et al, 2012;Feilke et al, 2016) indicate that this enzyme functions as a plastoquinol: oxygen oxidoreductase in vivo. For the dissipation of excess reducing equivalents, PTOX enzymes need to work in concert with chloroplast type II-NAD(P)H dehydrogenases.…”

Use of Bioinformatics Tools in Different Spheres of Life Sciences

“…For example, prior to the expression of an algal membrane protein, plastid terminal oxidase 1 (PTOX1), in tobacco chloroplasts, authors conducted a phylogenetic analysis to construct the evolutionary history and determine essential features of that particular polypeptide [30]. The phylogenetic analysis revealed that the Chlamydomonas reinhardtii PTOX1 (Cr-PTOX1) has typical signatures of higher plant PTOX such as iron-binding sites, a conserved exon and various blocks of amino acids to act as plastoquinol terminal oxidase [30]. Similarly, Chen et al [31] used phylogenetic analysis to study the evolutionary history of respiratory mechanisms in the deep-sea bacterium Shewanella piezotolerans WP3 [31].…”