Figure 1.

Mutant alleles of ZmWhy1. (A) Positions of Mu transposon insertions in the ZmWhy1 gene. Protein coding regions are indicated by rectangles, untranslated regions and introns by lines and Mu transposon insertions by triangles. The sequence of each insertion site is shown below, with the nine nucleotides that were duplicated during insertion underlined. The identity of the member of the Mu family is shown for each insertion (why1-2: Mu1/1.7; why1-1: MuDR), and was inferred from polymorphisms in the terminal inverted repeats. (B) Phenotypes of ZmWhy1 mutant seedlings grown for nine days in soil. Seedlings shown are homozygous for either the Zmwhy1-1 or Zmwhy1-2 allele, or are the heteroallelic progeny of a complementation cross. (C) Immunoblot showing loss of ZmWHY1 in mutant leaf tissue. Total leaf extract (10 µg protein, or dilutions as indicated) were analyzed. The same blot stained with Ponceau S is shown below, with the large subunit of Rubisco (RbcL) marked. hcf7 and iojap are pale green and albino maize mutants with weak and severe plastid ribosome deficiencies, respectively (25,26). The apparently higher levels of ZmWHY1 in Zmwhy1-1 mutants relative to Zmwhy1-2 mutants may be an artifact of the fact that samples were loaded on the basis of equal total protein: the abundant photosynthetic enzyme complexes make up the bulk of the protein in the Zmwhy1-2 extract but are missing in the Zmwhy1-1 extract, causing other proteins to appear over-represented. (D) RNA-dependent coimmunoprecipitation of ZmWHY1 with CRS1. Prior to immunoprecipitation, stroma was treated with DNAse or RNAse, or incubated under similar conditions without added nuclease (Mock). The stroma was then subjected to immunoprecipitation with the antibody named at top. Presence of CRS1 in the immunoprecipitation pellets was tested by immunoblot analysis with CRS1 antibody.