Figure 2.

Identification of the fug1 Alleles and RNAi Suppression Analysis.

(A) Schematic representation of the gene At1g17220, which corresponds to FUG1 (also shown as cpIF2). Locations of T-DNA insertions in two lines (fug1-2 and fug1-3) as determined by sequence analysis. Insertion sites are indicated by white arrowheads with the nucleotide positions (+2517 and −367, respectively; +1 corresponds to A of the initiation codon ATG in the genomic sequence). An amino acid substitution found in fug1-1 (+4437, L963F) is indicated by an asterisk. The region corresponding to a DNA fragment that was used for the RNAi suppression experiment is indicated by a red bar.

(B) Dissected young siliques from fug1-2 showing embryo lethality. Defective seeds detected in young siliques of fug1-2 are indicated by asterisks. Bars = 0.5 mm.

(C) Suppression of the var2 leaf variegation by fug1-3. Photographs of 5-week-old var2-1 (left) and var2-1 fug1-3 (right) are shown. The genotype of the var2-1 fug1-3 plants was determined by PCR with gene-specific primers (see Methods). Bars = 5 mm.

(D) Accumulation of FUG1 mRNA examined by RT-PCR. Primers for FtsH2(VAR2)+FtsH8, FUG1, and ACTIN2 were prepared, and RT-PCR was performed with total RNAs from 3-week-old plants as indicated above the gel. For each gene, two gels with different amplification cycles (shown on the right) are indicated. RT-PCR was repeated at least three times, and the representative result is shown.

(E) Accumulation of cpIF2 protein examined by immunoblot analysis. Total protein extracts from cotyledons of Col, var2-1, var2-1 fug1-1, fug1-1, and fug1-3 were separated by 7.5% SDS-PAGE and probed with anti-FUG1 (top), anti-FtsH2 (middle), and Lhcb1 (bottom). Samples were equally loaded based on total chlorophyll contents. We repeated immunoblot analysis at least three times, and the representative result is shown. The band of 105 kD corresponding to the mature size of cpIF2 is indicated by an open arrowhead. An E. coli extract from the strain carrying pTrc99AcpIF2Δ64 (see Figure 4) also detected the band of 105 kD (right). Additional bands detected by the E. coli extract (indicated by closed arrowheads) likely represent degradation products of cpIF2, since none of the signals were detected by cell extracts without expressing cpIF2 (data not shown).

(F) RNAi suppression of cpIF2 restores the formation of green sectors in true leaves. Schematic representation of the gene cassette used in RNAi (cpIF2-RNAi) is indicated on the top, consisting of the CaMV 35S promoter (Pro_35S), part of β-glucuronidase gene (ΔGUS), and the nopaline synthase terminator (NOS). The DNA fragment specific for FUG1 inserted in the gene cassette (shown in [A]) is highlighted by two red arrows (Antisense and Sense). Photographs of a parental var2-1 (var2-1) and two cpIF2-RNAi transgenics (transformants) displaying enhanced green sectors are indicated on the bottom. Bars = 1 mm.