Arabidopsis ANGULATA10 is required for thylakoid biogenesis and mesophyll development

Abstract

The chloroplasts of land plants contain internal membrane systems, the thylakoids, which are arranged in stacks called grana. Because grana have not been found in Cyanobacteria, the evolutionary origin of genes controlling the structural and functional diversification of thylakoidal membranes in land plants remains unclear. The angulata10-1 (anu10-1) mutant, which exhibits pale-green rosettes, reduced growth, and deficient leaf lateral expansion, resulting in the presence of prominent marginal teeth, was isolated. Palisade cells in anu10-1 are larger and less packed than in the wild type, giving rise to large intercellular spaces. The ANU10 gene encodes a protein of unknown function that localizes to both chloroplasts and amyloplasts. In chloroplasts, ANU10 associates with thylakoidal membranes. Mutant anu10-1 chloroplasts accumulate H2O2, and have reduced levels of chlorophyll and carotenoids. Moreover, these chloroplasts are small and abnormally shaped, thylakoidal membranes are less abundant, and their grana are absent due to impaired thylakoid stacking in the anu10-1 mutant. Because the trimeric light-harvesting complex II (LHCII) has been reported to be required for thylakoid stacking, its levels were determined in anu10-1 thylakoids and they were found to be reduced. Together, the data point to a requirement for ANU10 for chloroplast and mesophyll development.

Keywords: Arabidopsis thaliana; LHCII trimers; chloroplast; grana; mesophyll development; thylakoid biogenesis; thylakoid stacking..

Fig. 1.

Phenotypic characterization and rescue of the anu10-1 mutant. (A–C) Rosettes, (D–F) first-node leaves, and (G–I) bright-field micrographs of the subepidermal layer of palisade mesophyll cells from (A, D, G) the Ler wild type, (B, E, H) the anu10-1 mutant, and (C, F, I) a transgenic anu10-1 35Spro:ANU10 plant. (J) Adult plants. Pictures were taken (A–I) 16 and (J) 42 das. Scale bars indicate (A–F) 2mm, (G–I) 30 μm, and (J) 5cm. (K) Dry weight and (L) pigment content in Ler, anu10-1, and transgenic anu10-1 35Spro:ANU10 plants. Error bars indicate standard deviations. Asterisks indicate values significantly different from Ler in a Mann–Whitney U-test [(K) P<0.01, n=8 and (L) P<0.05, n=4].

Fig. 2.

Morphometry of anu10-1 mesophyll cells. (A) Distribution of palisade mesophyll cell area in first- and third-node leaves from Ler, anu10-1, and anu10-1 35S _pro :ANU10 plants (n=8). (B–G) Representative diagrams of the subepidermal layer of palisade mesophyll cells from (B–D) first- and (E–G) third-node leaves. Diagrams were drawn from differential interference contrast pictures taken from cleared leaves. (H–J) First-node leaf transverse sections from (H) Ler, (I) anu10-1, and (J) anu10-1 35S _pro :ANU10 plants. ad, adaxial surface, ab, abaxial surface. Scale bars indicate (B–G) 50 μm and (H–J) 500 μm. (K) Percentage of leaf transect area occupied by epidermis, mesophyll (including palisade and spongy mesophyll cells and bundle sheath cells), or air spaces in Ler (green), anu10-1 (red), and anu10-1 35S _pro :ANU10 (blue) first-node leaves. Error bars indicate standard deviations. Asterisks indicate values significantly different from Ler in a Mann–Whitney U-test (P<0.01, n=6).

Fig. 3.

Positional cloning of ANU10. (A) A mapping population of 332 F₂ plants derived from an anu10-1×Col-0 cross allowed a candidate region of 72kb to be defined in chromosome 1. Names and physical map positions of the molecular markers used for linkage analysis are shown. The number of recombinant chromosomes found and the total number of chromosomes analysed are indicated in parentheses. (B) Structure of the ANU10 gene with indication of the nature and position of the anu10 mutations. Boxes and lines indicate exons and introns, respectively. A white box represents the 3′-UTR. Triangles indicate T-DNA insertions.

Fig. 4.

Visualization of ANU10 _pro :GUS activity in a wild-type background. (A) Seedling, (B) detail of the shoot apex, (C) roots, and (D) cotyledon. (E–H) Vegetative leaves from the (E) first, (F) third, (G) fifth, and (H) seventh nodes. (I) Inflorescence, (J) young cauline leaf, (K) immature flowers, (L) mature flower, and (M) immature and (N) mature siliques. Pictures were taken (A) 3, (B) 6, (C–H) 13, and (I–N) 42 das. Scale bars indicate (A, D–G, I, J, L–N) 1mm, (C, H, K) 500 μm, and (B) 100 μm.

Fig. 5.

Subcellular and suborganellar localization of ANU10. (A–F) Confocal micrographs of the subepidermal layer of palisade mesophyll cells from (A–C) Ler and (D–F) anu10-1 35S _pro :ANU10:GFP transgenic plants. Micrographs show (A, D) the chlorophyll autofluorescence of the chloroplasts, (B, E) the GFP fluorescence, and (C, F) an overlay of the chlorophyll and GFP signals, showing their co-localization in (F). Pictures were taken from first-node leaves collected 16 das. Scale bars indicate 50 μm. (G) Western blot analysis of the proteins in chloroplast, stroma, and thylakoid fractions isolated from anu10-1 35S _pro :ANU10:GFP transgenic plants collected 16 das. Primary antibodies against GFP, the large Rubisco subunit (RbcL), and the PsbO subunit of PSII were used. Molecular mass markers are indicated on the right.

Fig. 6.

Localization of the ANU10 protein and effect of anu10 mutations on root growth. (A, B) Confocal micrographs of the root apex from an anu10-1 ANU10 _pro :ANU10:GFP transgenic plant: (A) differential interference contrast (DIC) image and (B) an overlay of the GFP fluorescence and the DIC image. (C) Root apex from a Ler plant stained with lugol. Scale bars indicate 30 μm. (D) Main root length of anu10-1, anu10-2, and anu10-3 mutants, their respective wild types, and anu10-1 35S _pro :ANU10 transgenic plants. Error bars indicate standard deviations. Asterisks indicate values significantly different from the wild type in a t-test (P<0.05, n=30).

Fig. 7.

Ultrastructure of anu10-1 chloroplasts. Transmission electron micrographs of palisade mesophyll cell chloroplasts from (A–C) Ler, (D–F) anu10-1, and (G–I) anu10-1 35S _pro :ANU10. Arrowheads in (F) indicate unstacked thylakoid membranes in the anu10-1 mutant. Pictures were taken from first-node leaves collected 16 das. Scale bars indicate (A, D, G) 5 μm, (B, H) 2 μm, (E) 1 μm, and (C, F, I) 200nm. (J–M) Comparison of (J) the diameter (x-axis) and (K) height (y-axis) of granal stacks, (L) the number of membrane layers in granal stacks, and (M) the number of stacks in chloroplast sections from Ler, anu10-1, and anu10-1 35S _pro :ANU10. The number of grana per chloroplast section was determined from images of individual chroroplasts similar to those shown in B, E, and H. Asterisks indicate values significantly different from the wild type in a t-test (P<0.05, n=10–20).

Fig. 8.

Thylakoidal protein complexes in the anu10 mutants. Blue native PAGE of photosynthetic protein complexes from anu10-1, anu10-2, and anu10-3 mutants, their respective wild types, and anu10-1 35S _pro :ANU10 and anu10-1 35S _pro :ANU10:GFP transgenic plants. PSII, photosystem II; PSI, photosystem I; Cyt b ₆ f, cytochrome b ₆ f complex; LHCII, light-harvesting chlorophyll a/b-binding protein complex II.

Fig. 9.

Expression of nuclear and plastid genes in the anu10-1 mutant. (A, B) qRT-PCR analysis of the expression of (A) LHCB1, LHCB2, LHCB3, LHCB5, HEMA1, and ORE1 nuclear genes, and (B) accD, psbA, rbcL, rrn16, rrn23, and atpB plastid genes in Ler and anu10-1 rosettes collected 16 das. Bars indicate the relative expression levels, determined by the comparative C_T method, and normalized with the expression of the 18S rRNA housekeeping gene. Error bars indicate the interval delimited by 2^{–(ΔΔCT± SD)}. Asterisks indicate ΔC_T values significantly different from those of Ler in a Mann–Whitney U-test (P<0.01; n=9).