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. 2021 Apr 30;12:676036. doi: 10.3389/fpls.2021.676036

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Copyright © 2021 Chang, Li, Cui, Li, Song, Chang, Fu, Wang, Huang, Luo, Shan, Wang, Wang, Xu and Fu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Ectopic expression of CtpA from lower photosynthetic organisms in the atctpa mutant. (A) Schematic diagrams of fusion proteins SeCtpA-HA (from Synechococcus elongatus), CrCtpA-HA (from Chlamydomonas reinhardtii), and PpCtpA-HA (from Physcomitrella patens). AtCtpA-TP is the chloroplast TP of AtCtpA protein; mPpCtpA, mCrCtpA, and mSeCtpA are the mature proteins; 2HA indicates a double hemagglutinin tag. (B) Phenotype of atctpa plants transformed with CaMV 35S promoter-driven SeCtpA-HA, CrCtpA-HA, and PpCtpA-HA. Plants were grown on 1/2 MS medium with 1% sucrose under continuous LL light (25 μmol/s/m²) for 3 weeks. At least five transgenic lines per construct were examined. Scale bars represent 1 cm. (C) Chloroplast sub-compartment distribution of AtCtpA, SeCtpA, CrCtpA, and PpCtpA. Intact chloroplasts from transgenic plants expressing AtCtpA-HA, SeCtpA-HA, CrCtpA-HA, and PpCtpA-HA were fractionated into stroma (S), thylakoid membrane (M), and thylakoid lumen (L). The distribution of CtpA in the three fractions was examined by immunoblotting with a-HA and compared with the thylakoid lumen protein PC, the thylakoid membrane protein D1, and the stromal protein ClpC. (D) D1 processing status in atctpa plants transformed with CaMV 35S promoter-driven SeCtpA-HA, CrCtpA-HA, and PpCtpA-HA. D1 processing in plants in panel (B) was analyzed by immunoblotting using the following antibodies: α-AtCtpA for AtCtpA, α-HA for ectopically expressed CtpA-HA, α-D1 for D1 and pD1, and α-pD1 tail for pD1. The filters stained with Ponceau S (Pon. S) were used as loading control. (E) BN-PAGE analyses of atctpa plants transformed with CaMV 35S promoter-driven SeCtpA-HA, CrCtpA-HA, and PpCtpA-HA. The transgenic and WT plants were grown in soil under continuous NL light (80 μmol/s/m²) for 3 weeks before analyses. Thylakoid membrane protein complexes (12 μg chlorophyll) were loaded on a BN-PAGE gel for electrophoresis.