Chloroplast protein and pigment defects in tha8 mutants. (A) Visual phenotype of a tha8 mutant and normal sibling (WT) grown for 8 d in soil. (B) Western blot analysis of thylakoid proteins in tha8 mutants. Proteins were extracted from leaves of wild-type and tha8 seedlings and analyzed on replicate immunoblots by probing with antibodies to AtpA (α subunit of ATP synthase), Cyt f (subunit of cytochrome b6f complex), D1 (reaction center protein of photosystem II), PSA-D (core subunit of photosystem I), or LHCP (major light harvesting chlorophyll binding protein of PSII). An example of a Ponceau S stained filter is shown below, with RbcL (large subunit of Rubisco) marked. (C) Aberrant metabolism of Tat substrates in tha8 mutants. Mature (m) and incompletely processed stromal intermediates (i) are marked. (D) iOE23 and iOE16 in tha8 mutants accumulate outside of the thylakoid membrane. Mutant chloroplasts (cp) were separated into stroma and thylakoids. The thylakoids were treated with thermolysin (therm) with or without the addition of Triton X-100. Analogous results were obtained with iOE16 (data not shown). (E) Positions of the Mu insertions in tha8 mutants. The tha8 gene lacks introns. The approximate position of the transit peptide (TP) cleavage site is marked. The sequences flanking the insertions are shown below, with the 9-bp target site duplications underlined. (F) Predicted structure of THA8. This structure prediction was made via the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) using the sequence of mature maize THA8 (i.e., lacking the inferred transit peptide). The arrow marks the beginning of the canonical PPR tract.
