Microarray analysis confirms the specificity of a Chlamydomonas reinhardtii chloroplast RNA stability mutant

Abstract

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687-697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

Figure 1.

Differential RNA accumulation in mcd1-1, mcd1-2, and mcd1-2/mcd2-1. A, Representative fluorescent images of three replicate spots for the indicated genes from a single microarray experiment. Test cDNAs (mcd1-1, mcd1-2, and mcd1-2/mcd2-1) were labeled with Cy5 (red), while the reference cDNAs (wild type [WT] and mcd1-2 when compared to the suppressor) were labeled with Cy3 (green). B, RNA gel blots were used to test for petD, atpA, cemA, psbA, and nad2 mRNAs. Ethidium bromide-stained 25S rRNA is the loading control. Polycistronic transcripts for atpA and cemA are indicted and numbers correspond to polycistronic transcripts in Figure 3A. C, Hierarchical clustering of expression ratio data for the two mcd1 mutants (mcd1-1, mcd1-1 versus wild type; mcd1-2, mcd1-2 versus wild type). Ratio data are averages from three independent hybridizations. Green represents a decrease in RNA abundance in the mutant strains and red represents an increase in RNA abundance. The color key for log2 fold changes in expression (−3 to +3) is shown. Gene names are shown at the right, and arrows indicate RNAs tested in B. Vertical lines marked 1 and 2 highlight RNAs discussed in the text.

Figure 2.

Scatter plots of the three microarray experiments. Each point indicates the average fluorescent of test cDNA (y axes) plotted against the reference cDNA (x axes) in log scale. Diagonal lines 10 and 2 indicate the 10-fold and 2-fold increase cut-off lines, and −10 and −2 diagonal lines indicate the 10-fold and 2-fold decrease cut-off lines. The diagonal line 1 indicates the ratio of 1.0 with no change in RNA between two cells. Selected RNAs with significant differences between strains are labeled.

Figure 3.

RNA gel blots and immunoblots to test accumulation of transcripts and proteins from the chloroplast atpA/psbI/cemA/atpH gene cluster. A, Diagram of the eight transcripts produced from this gene cluster as previously reported (Drapier et al., 1998). Filled boxes are gene coding regions, and numbered heavy arrows represent known transcripts with the corresponding coding regions. Small bent arrows show the known promoters. Asterisks indicate those atpA-containing transcripts that are reduced in mda1-2-containing strains. B, RNA gel blots were probed for chloroplast cemA and psbA mRNAs with ethidium bromide-stained 25S rRNA as the loading control. Transcripts labeled at right correspond to those labeled in A. C, Immunoblots testing accumulation of chloroplast SUIV protein and the ATP synthase α- and β-subunits. Proteins were visualized by enhanced chemiluminescence.

Figure 4.

RNA gel blots of parents and representative progeny from genetic crosses. A, Parents and tetrad numbers 43 and 21 from the cross shown at the top were subjected to RNA gel-blot analysis for atpA, cemA, petD, and psbA chloroplast mRNAs, with ethidium bromide-stained 25S rRNA as a loading control. Numbered lanes (1–4) correspond to four progeny within a tetrad, and the inferred genotype of each progeny is shown at bottom, along with photosynthetic phenotypes (+ or −). B, Parents, tetrad numbers 2 and 3 progeny, and a wild-type control for the cross shown at the top were tested for atpA and petD mRNAs, with ethidium bromide-stained 25S rRNA as a loading control.