Arabidopsis MDA1, a nuclear-encoded protein, functions in chloroplast development and abiotic stress responses

Abstract

Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription termination factor (mTERF) family in Arabidopsis thaliana allowed us to identify 75 lines carrying T-DNA insertions. Two of them were homozygous for insertions in the At4g14605 gene, which we dubbed MDA1 (MTERF DEFECTIVE IN Arabidopsis1). The mda1 mutants exhibited altered chloroplast morphology and plant growth, and reduced pigmentation of cotyledons, leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses during seedling establishment, possibly as a result of reduced ABA sensitivity. Loss of MDA1 function caused up-regulation of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast gene transcripts. Double mutant analyses indicated that MDA1 and the previously described mTERF genes SOLDAT10 and RUG2 act in different pathways. Our findings reveal a new role for mTERF proteins in the response to abiotic stress, probably through perturbed ABA retrograde signalling resulting from a disruption in chloroplast homeostasis.

Figure 1. mTERF motifs in members of the mTERF family.

(A) Modular architecture of the Arabidopsis thaliana (At) mTERF proteins MDA1, BSM/RUG2 and SOLDAT10, human (Hs) MTERF3, Drosophila melanogaster (Dm) D-MTERF3 and Paracentrotus lividus (Pl) mtDBP. The diagram was drawn using SMART. mTERF motifs are shown as ellipses. The number of amino acids of each protein is indicated. (B) Sequence logo for the mTERF motifs of the above mentioned proteins. Numbers on the abscissa represent positions in mTERF motifs and the ordinates represent the information content measured in bits. The sequence logo was derived using WebLogo (http://weblogo.berkeley.edu/). (C) Multiple alignment of the amino acid sequence of part of the proteins encoded by the At MDA1, SOLDAT10 and BSM/RUG2, Arabidopsis lyrata Al915404, Oryza sativa Os02g39040.1, mouse [Mus musculus (Mm)] Mterf3, rat [Rattus norvegicus (Rn) Mterf3], Hs MTERF3, Dm D-MTERF3 and Pl mtDBP genes. Residues conserved across five or more sequences are shaded in black, and similar residues are shaded in grey. Numbers indicate amino acid positions. The alignment was obtained using ClustalX v1.5b. A continuous line indicates an mTERF motif in MDA1.

Figure 2. Some morphological traits of the mda1 mutants.

(A–C) Seven-day-old seedlings and (D–F) 21-day-old rosettes of mda1-1 (B, E), mda1-2 (C, F) and Col-0 (A, D). (G) Forty-five-day-old plants grown in soil. (H) Fourteen-day-old plants grown on vertically orientated agar plates. Bars  = 1 mm (A–H). (I) Concentration (mg/g of fresh weight) of chlorophyll a (Ca) and b (Cb), and carotenoids in the mda1 mutants and Col-0. Data represent mean of 10 samples of 15-day-old plants per genotype ± standard deviation (SD). Two asteriscs indicate that the value is significantly different from the wild type at P<0.01, respectively, using Student’s t-test.

Figure 3. Chloroplast structure in the mda1 mutants.

(A, D, G) Confocal micrographs showing chlorophyll autofluorescence in mesophyll cells of Col-0 (A), mda1-1 (D) and mda1-2 (G) third-node leaves. (B, C, E, F, H and I). Transmission electron micrographs of chloroplasts of a Col-0 (B), mda1-1 (E) and mda1-2 (H) mesophyll cell. Close-up views of Col-0 (C), mda1-1 (F) and mda1-2 (I) chloroplasts. Photographs were taken 21 days after stratification (das). Bars  = 20 µm (A, D, G), 5 µm (B, E, H) and 2 µm (C, F, I).

Figure 4. Effects of NaCl, KCl and mannitol on seedling establishment in the mda1 mutants.

(A, B) Col-0 (A) and mda1-1 (B) seedlings germinating on growth medium supplemented with 200 mM NaCl. The inset images correspond to magnifications of the seedlings indicated by arrows. The mutants display green expanded cotyledons, a phenotypic trait barely observed in Col-0. Scale bars: 1 mm. (C–E) Seedling establishment of the mda1 mutants and Col-0 in various NaCl (C), KCl (D) and mannitol (E) concentrations. We considered only those seedlings displaying green expanded cotyledons. Error bars represent SD. Each value corresponds to the average of two independent experiments with two to four replicates of 50–100 seeds each. Germination was scored at 10 das.

Figure 5. Tolerance to ABA and NaCl of the mda1 mutants.

(A) Effects of different ABA concentrations on seedling establishment in the mda1 mutants. Data are means of two independent experiments with three replicates of 50–100 seeds each scored 10 das. Error bars represent SD. The sañ5 (abi4-2) ABA insensitive and salt-tolerant mutant was used as a positive control . (B, C) Sensitivity to ABA and NaCl of mda1 plants. The individuals were transplanted 9 das from non-supplemented growth media to media supplemented with 0, 100 mM NaCl or 4 µM ABA. 12 days after the transfer, tolerance was estimated by determining the fresh weight and root length of the plants transferred to NaCl or ABA supplemented media and referring them to those of the same genotypes transferred to non-supplemented media. These values are represented as percentages of (B) fresh weight and (C) root length of plants transferred to non-supplemented media. Each value corresponds to the mean ± SD of the fresh weight or root length of 15 plants of each genotype. One and two asterisks indicate that the value is significantly different from the wild-type at P<0.05 or P<0.01, respectively, using Student’s t-test.

Figure 6. Genetic interactions between mTERF mutants.

(A) Additive phenotypes of mda1 rug2-2 and mda1 soldat10 double mutants. Rosettes from a wild-type (Col-0) and the single mutants rug2-2, mda1-1 and mda1-2 (in a Col-0 genetic background), soldat10 (in a Ler genetic background) and the mda1-1 rug2-2, mda1-1 soldat10, mda1-2 rug2-2 and mda1-2 soldat10 double mutants. (B) Genetic interaction between rug2-1 and soldat10. Rosettes from the rug2-1 and soldat10 single mutants, both in a Ler genetic background, and the rug2-1 soldat10 double mutant. Pictures were taken 21 das. Scale bars: 1 mm.