Expression strategies for the efficient synthesis of antimicrobial peptides in plastids.

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.

Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

MAIN CONCLUSION: AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

CRP1 Protein: (dis)similarities between Arabidopsis thaliana and Zea mays.

Biogenesis of chloroplasts in higher plants is initiated from proplastids, and involves a series of processes by which a plastid able to perform photosynthesis, to synthesize amino acids, lipids, and phytohormones is formed. All plastid protein complexes are composed of subunits encoded by the nucleus and chloroplast genomes, which require a coordinated gene expression to produce the correct concentrations of organellar proteins and to maintain organelle function. To achieve this, hundreds of nucleus-encoded factors are imported into the chloroplast to control plastid gene expression. Among these factors, members of the Pentatricopeptide Repeat (PPR) containing protein family have emerged as key regulators of the organellar post-transcriptional processing. PPR proteins represent a large family in plants, and the extent to which PPR functions are conserved between dicots and monocots deserves evaluation, in light of differences in photosynthetic metabolism (C3 vs. C4) and localization of chloroplast biogenesis (mesophyll vs. bundle sheath cells). In this work we investigated the role played in the process of chloroplast biogenesis by At5g42310, a member of the Arabidopsis PPR family which we here refer to as AtCRP1 (Chloroplast RNA Processing 1), providing a comparison with the orthologous ZmCRP1 protein from Zea mays. Loss-of-function atcrp1 mutants are characterized by yellow-albinotic cotyledons and leaves owing to defects in the accumulation of subunits of the thylakoid protein complexes. As in the case of ZmCRP1, AtCRP1 associates with the 5' UTRs of both psaC and, albeit very weakly, petA transcripts, indicating that the role of CRP1 as regulator of chloroplast protein synthesis has been conserved between maize and Arabidopsis. AtCRP1 also interacts with the petB-petD intergenic region and is required for the generation of petB and petD monocistronic RNAs. A similar role has been also attributed to ZmCRP1, although the direct interaction of ZmCRP1 with the petB-petD intergenic region has never been reported, which could indicate that AtCRP1 and ZmCRP1 differ, in part, in their plastid RNA targets.