The SCABRA3 nuclear gene encodes the plastid RpoTp RNA polymerase, which is required for chloroplast biogenesis and mesophyll cell proliferation in Arabidopsis

Abstract

In many plant species, a subset of the genes of the chloroplast genome is transcribed by RpoTp, a nuclear-encoded plastid-targeted RNA polymerase. Here, we describe the positional cloning of the SCABRA3 (SCA3) gene, which was found to encode RpoTp in Arabidopsis (Arabidopsis thaliana). We studied one weak (sca3-1) and two strong (sca3-2 and sca3-3) alleles of the SCA3 gene, the latter two showing severely impaired plant growth and reduced pigmentation of the cotyledons, leaves, stem, and sepals, all of which were pale green. The leaf surface was extremely crumpled in the sca3 mutants, although epidermal cell size and morphology were not perturbed, whereas the mesophyll cells were less densely packed and more irregular in shape than in the wild type. A significant reduction in the size, morphology, and number of chloroplasts was observed in homozygous sca3-2 individuals whose photoautotrophic growth was consequently perturbed. Microarray analysis showed that several hundred nuclear genes were differentially expressed in sca3-2 and the wild type, about one-fourth of which encoded chloroplast-targeted proteins. Quantitative reverse transcription-PCR analyses showed that the sca3-2 mutation alters the expression of the rpoB, rpoC1, clpP, and accD plastid genes and the SCA3 paralogs RpoTm and RpoTmp, which respectively encode nuclear-encoded mitochondrion or dually targeted RNA polymerases. Double-mutant analysis indicated that RpoTmp and SCA3 play redundant functions in plant development. Our findings support a role for plastids in leaf morphogenesis and indicate that RpoTp is required for mesophyll cell proliferation.

Figure 1.

Some morphological traits of the phenotype of sca3 mutants. Images correspond to cotyledons of 7-d-old seedlings (A–D), 21-d-old rosettes (E–H), and third node vegetative leaves (I–L) of the sca3-1 and sca3-2 mutants and their corresponding wild types (Ler and Col-0, respectively). Six-week-old plants (M) grown on soil and details of their siliques (N and O) and inflorescences (P and Q). R, Fifteen-day-old plants vertically grown on agar medium. All plants were homozygous for the mutations shown. Scale bars indicate 1 mm (A–D, H–L, and N–Q), 5 mm (E–G), 10 mm (R), and 25 mm (M).

Figure 2.

Positional cloning and structural analysis of the SCA3 gene. A, Map-based strategy followed to identify the SCA3 gene. After studying 882 chromosomes, a total of 33 recombinant events (in parentheses) were identified in a region of 11.7 cm on chromosome 2, flanked by the PLS8 and nga1126 markers. A candidate interval of 70 kb was finally delimited, flanked by the CER460106 and CER429871 markers and encompassing the T29E15 and F27D4 bacterial artificial chromosome clones, which included the At2g24060 to At2g24200 annotated genes. B, Structure of the SCA3 gene, with indication of the position and nature of the sca3 mutations. Exons and introns are indicated by black boxes and lines, respectively. Triangles indicate T-DNA insertions. Horizontal arrows indicate the oligonucleotides used to characterize the structure and expression of SCA3, which are not drawn to scale. C, Alignment of the sixteenth exon-intron junction region of the SCA3 gene in the wild-type Ler and the sca3-1 mutant. The sequences shown correspond to the single splicing pattern found in the wild-type SCA3 allele (Ler) and the two detected in the sca3-1 mutant (here named sca3-1a and sca3-1b). Upper- and lowercase letters indicate exonic and intronic sequences, respectively. The eight-nucleotide segment absent from sca3-1b mRNA is underlined. An arrowhead indicates the single-nucleotide change found in sca3-1 genomic DNA. The cryptic splicing donor site within exon 16 used in sca3-1b is boxed. D, Alignment of the amino acid sequences corresponding to the C-terminal part of the protein products of the wild-type SCA3 and the mutant sca3-1 alleles of the SCA3 gene. An asterisk denotes a stop codon. E, Expression analysis of the SCA3 gene by RT-PCR in sca3/sca3 and wild-type individuals. The bands shown were obtained after PCR amplifications were performed using as a template genomic DNA (gDNA) or RNA extracted from 3-week-old plants and reverse transcribed, and the primers shown. The OTC gene was used as an internal control. F, Expression analysis of the SCA3 gene by RT-PCR in sca3-2/sca3-2 and Col-0 individuals. The bands shown were obtained after PCR amplifications performed using as a template gDNA or RNA extracted from 3-week-old plants and reverse transcribed, and the primers shown. G, Alignment of the amino acid sequences corresponding to the protein products of the wild-type SCA3 and the mutant sca3-2 alleles of the SCA3 gene. An asterisk denotes a stop codon.

Figure 3.

Predicted amino acid sequence of the Arabidopsis SCA3 protein (RpoTp; Y08463). Residues shaded in black are identical across the sequences of homologous gene products from H. vulgare (CAD45446), T. aestivum (AF091839), Z. mays (AF127022), O. sativa (AB120430), N. sylvestris (AJ302020), and tobacco (AJ416570). Residues with similar chemical properties conserved across all seven sequences are shaded gray. The alignment was obtained using ClustalX, version 1.5b. Continuous and dotted thin lines indicate the domains (I–XI) and the C-motif that are conserved among T7 phage-type NEP plastid RNAPs, respectively (McAllister and Raskin, 1993; Chang et al., 1999). The amino acid sequences deleted in the mutant protein products of sca3-1b (see Fig. 2), sca3-2, and sca3-3 are indicated by white boxes, thick discontinuous lines, and black boxes, respectively.

Figure 4.

Scanning electron micrographs of sca3/sca3 leaves. Micrographs are shown of the adaxial surface (A–D) of whole leaves and details of their adaxial (E–H) and abaxial epidermises (I–L). All plants were homozygous for the mutations shown. Leaves were collected 28 d after sowing. Scale bars indicate 1 mm (A–D) and 10 μm (E–L).

Figure 5.

Reduced cell density in the mesophyll of sca3/sca3 leaves. A to H, Confocal micrographs, showing fluorescing chlorophyll within mesophyll cells of whole third leaves (A–D), and details of the mesophyll (E–H). Transverse sections of leaves (I–L) and whole leaves (M–P). All plants were homozygous for the mutations shown. Leaves were collected 28 d after sowing. Scale bars indicate 1 mm (A–D), 50 μm (E–L), and 0.5 mm (M–P).

Figure 6.

Transmission electron micrographs of sca3/sca3 chloroplasts. A to C, Representative chloroplasts of Col-0 and the sca mutants. Chloroplasts of Ler and Col-0 were indistinguishable and no differences were found between those of their mesophyll cells and those of cells neighboring the midvein. D, One of the chloroplasts occasionally seen in sca3-2/sca3-2 mesophyll cells, showing enlarged thylakoids (arrows) and vacuoles (v). E, Chloroplasts of a cell adjacent to the midvein of the sca3-2/sca3-2 mutant. All plants were homozygous for the mutations shown. All leaves were collected from the third node 21 d after sowing. Scale bars indicate 1.5 μm (A and B), 0.6 μm (C and D), and 1.2 μm (E).

Figure 7.

Genetic interaction between the SCA3 and RpoTmp genes. sca3-2 (A), rpoT;2 single mutants (B), and sca3-2 rpoT;2 double mutants (C and D). Images were taken 21 d after sowing. Scale bars indicate 1 mm.