Figure 1.
The tetrapyrrole biosynthetic pathway in plants.
Enzymes (in bold capital letters) and intermediates of the nine steps of the common part of the pathway as well as the four end-products (in italics) are shown. GTS: Glutamyl-tRNA synthase; GluTR: Glutamyl-tRNA reductase; GSA-AT: Glutamate-1-semialdehyde amino-transferase; ALAD: 5-aminolaevulinic acid-dehydratase; PBGD: porphobilinogen deaminase; UROS: Uroporphyrinogen III synthase; UROD: Uroporphyrinogen III decarboxylase; CPO: coproporphyrinogen III oxidase; PPO: protoporphyrinogen III oxidase. Asterisks indicate genes for which a mutant phenotype has been reported in Arabidopsis. Redrawn from [23].
Figure 2.
Lesion phenotype in the rug1 mutant.
(a, d) Three-week-old rosettes of the rug1 mutant and the wild-type Ler. (b, e) Close-up views of third-node vegetative leaves from the plants shown in panels (a) and (d). (c, f, h, i) Confocal micrographs showing fluorescing chlorophyll within mesophyll cells of (c, f) whole third-node leaves [those shown in (b) and (e)] and (h) details of the subepidermal layer of mesophyll cells of Ler and (i) the boundary between a green and a pale sector in a rug1 leaf. (g) 45-day-old plants grown in soil. (j, k) Transverse sections of third leaves. Bars = (a–f) 1 mm, (g) 1 cm, (h, i) 250 µm, and (j, k) 50 µm.
Figure 3.
Scanning electron micrographs of rug1 leaves.
(a, c) Adaxial surface of third-node leaves and (b, d–f) details of the adaxial epidermis. (e, f) Different magnifications of the area boxed in red in (c), which corresponds to a necrotic sector. Pictures were taken 21 das (days after stratification). Bars = (a, c) 1 mm, (b, d, e) 100 µm, and (f) 10 µm.
Figure 4.
(a–c) 21-day-old rosettes of (a) Ler and (b) rug1, stained with toluidine blue, and (c) a third-node leaf from the plant shown in (b). Arrows in (c) indicate defective cuticle in a necrotic sector. (d–g) Trypan blue staining of (d) Ler and (f) rug1 third-node leaves and (e, g) close-up views of the leaves shown in (d) and (f), revealing dead cells in rug1. (h–o) (h, j, l, n) Rosettes of the genotypes indicated and (i, k, m, o) visualization of H2O2 accumulation by means of DAB staining of (i, k, m) one or (o) all of their leaves. Plants were grown under (a–k) continuous light or (l–o) long day conditions (16-h light/8-h dark). Bars = (a, b, h, j, l) 5 mm, (c, d, f, i, k, m–o) 1 mm, and (e, g) 200 µm.
Figure 5.
Conservation of PBGD and structure of the RUG1 gene.
Alignment of the predicted amino acids of the Arabidopsis RUG1 (NP_196445) protein with those of its putative orthologues from Pisum sativum (Q43082), Triticum aestivum (AAL12220), Oryza sativa (NP_001046017), Escherichia coli (YP_001460596), Homo sapiens (NP_000181), Mus musculus (AAH03861), Danio rerio (NP_957448) and Saccharomyces cerevisiae (NP_010076). Residues identical across all the sequences are shaded black; residues with similar chemical properties conserved across all five sequences are shaded grey. Numbers correspond to amino acid positions. Continuous lines indicate the N-terminal chloroplast transit peptide (as identified by the TargetP v1.0 program; [82]; http://www.cbs.dtu.dk/services/TargetP/). The alignment was obtained using ClustalX v 1.5b. The highly conserved amino acid that is changed in the rug1 mutant is indicated by an asterisk. A schematic representation of the RUG1 gene is also shown, with indication of the position of the rug1 mutation. Exons and introns are represented by boxes and lines, respectively. White boxes correspond to the 5′ and 3′ untranslated regions. The predicted translation start (ATG) and stop (TGA) codons are indicated. Horizontal arrows, not drawn to scale, indicate the oligonucleotides used to characterize the structure of RUG1.
Figure 6.
Measurements of PBGD activity and accumulation of PBG in rug1 and Ler.
(a) PBGD activity in enzyme units per milligram of protein and (b) PBG accumulation in micrograms per gram of fresh weight in Ler and rug1 plants grown under long day conditions (16-h light/8-h dark) or continuous light. Asterisks indicate rug1 values significantly different from those of the wild type (Students t-test, P<0.01).