Article Figures & Data
Figures
- Figure 1.
Identification of a mutant sdh1-1 allele and molecular characterization of heterozygous mutant plants. A, Genomic organization of the SDH1-1 gene. Exons are presented as black boxes. The T-DNA insertion site is indicated. Horizontal arrows show the position of primers. The T-DNA insert is not drawn to scale. LB, T-DNA left border; P, genomic PstI sites flanking the T-DNA insert. B, Identification of heterozygous mutant plants. Genotyping was performed by PCR amplification with primers 1 and 2 to identify the mutant allele (m), and primers 2 and 3 to identify the wild-type allele (w). Six (1–6) heterozygous mutant plants were found. wt lanes correspond to a control with DNA from a wild-type plant, and lane −DNA to a PCR control without template. C, Northern-blot analysis of SDH1-1 expression in five heterozygous mutant plants (lanes 1–5) and three wild-type plants (lanes wt). Each lane was loaded with 15 μg of total RNA isolated from flowers. The blot was hybridized with specific SDH1-1 probe (derived from the 3′ UTR) and then with an actin probe as loading control. The SDH1-1 transcript was present at a lower level in heterozygous mutant plants. D, Complex II activity is reduced in heterozygous mutant plants. SQR activity of wild-type and heterozygous mutant seedlings was determined in the presence or absence of thenoyltrifluoroacetone (TTFA), a complex II inhibitor. Three independent experiments were performed and the actual values were 17.8, 14.6, and 15.3 nmol of reduced DCIP min−1 mg−1 of protein for wild type seedlings, and 12.0, 10.5, and 10.1 nmol of reduced DCIP min−1 mg−1 of protein for mutant seedlings. Error bars correspond to sds.
- Figure 2.
SDH1-1/sdh1-1 plants contain a unique complex T-DNA insertion. Southern-blot analysis was performed to characterize the insertion. Total DNA (6 μg) from wt (w) or mutant (m) plants was digested with PstI and hybridized sequentially with probes directed to the T-DNA (GUS probe), the SDH1-1 upstream region (probe 1), and the SDH1-1 downstream region (probe 2). The presence of a new PstI restriction site (P) was inferred from the hybridization results and is shown in gray, in a sequence of unknown origin.
- Figure 3.
Reproductive abnormalities in SDH1-1/sdh1-1 mutant plants. A, Mature pollen grains from a wild-type plant. Bar = 20 μm. B, Mature pollen grains from a heterozygous mutant plant. A substantial proportion of pollen grains are collapsed (arrowheads). Bar = 20 μm. C, Mature siliques of a SDH1-1/sdh1-1 plant are shorter and contain less seeds than mature siliques of the wild type. Bar = 2 mm. D, Closeup of a developing SDH1-1/sdh1-1 silique. Arrowheads denote aborted, white, and shrunken ovules. A wild-type seed is indicated by an asterisk. E, Seed set is reduced in SDH1-1/sdh1-1 siliques. Seed set per silique was scored along the main inflorescence axis for six T3 Kanr SDH1-1/sdh1-1 plants and two wild-type plants. Silique 1 (first, older flower) to silique 16 were grouped in four groups of four siliques for each plant. Total counts of 28.2 and 18.9 seeds/silique were obtained for wild type and SDH1-1/sdh1-1 plants, respectively (last two bars). Error bars are sds.
- Figure 4.
Loss of SDH1 function affects pollen viability. Alexander's staining was performed with wild type (A and C) and heterozygous SDH1-1/sdh1-1 mutant (B and D) anthers. In these bright-field images, wild-type, viable pollen grains show intensive purple staining in the cytoplasm, whereas dead, unviable pollen grains are devoid of cytoplasm and exhibit only the green staining of the exine outer layer. Bars = 10 μm.
- Figure 5.
Pollen development in SDH1-1/sdh1-1 mutant plants. Anther sections were stained with Toluidine blue and photographed by bright-field microscopy. Abnormal pollen grains are indicated by arrowheads. Magnification is the same in all micrographs (Bars = 20 μm). GN, Generative cell nucleus; N, nucleus; Tp, tapetum; V, vacuole; VN, vegetative cell nucleus. A, Anther from the wild type at the vacuolated microspore stage. B, Anther from the SDH1-1/sdh1-1 mutant, at the vacuolated microspore stage. All microspores are indistinguishable from those of wild-type anthers. C, Anther from the SDH1-1/sdh1-1 mutant after pollen mitosis I, showing normal pollen grains with two nuclei, and abnormal pollen grains (arrowheads) not entering mitosis. D, Anther from the SDH1-1/sdh1-1 mutant after pollen mitosis II, showing normal pollen grains with a dense cytoplasm, and abnormal pollen grains (arrowheads) containing cell debris. E, Anther just before dehiscence, containing mature pollen grains and collapsed pollen grains devoid of cytoplasm (arrowheads).
- Figure 6.
Transmission electron microscopy analysis of anthers in the SDH1-1/sdh1-1 mutant. Ex, Exine layer; GN, generative nucleus; L, lipid body; M, mitochondrion; N, nucleus; Nu, nucleolus; S, starch granules within plastids; V, vacuole; VN, vegetative nucleus. Bar = 5 μm in A, C to E, and G to I; bar = 1 μm in B, F, and J. A, Microspores at the vacuolated stage (anther stage 9 according to Sanders et al., 1999). All microspores had wild-type morphology, with numerous mitochondria, starch granules within plastids, and a normal exine outer layer. B, Closeup of the microspore shown in A. C, Bicellular pollen grain from SDH1-1/sdh1-1 anthers. The asymmetric first mitotic division has occurred (early anther stage 11). D, Same anther as in C. In addition to pollen with wild-type morphology (C), abnormal pollen grains were present. They did not undergo mitosis and cell content detached from the outer layers. E and F, Later developmental stage of wild-type pollen grains from SDH1-1/sdh1-1 anthers. G, Mature wild-type pollen grain from SDH1-1/sdh1-1 anthers. H and I, Later developmental stages of aberrant pollen grains from SDH1-1/sdh1-1 anthers. Cellular structures and content progressively disappeared and internal structures were no longer distinguishable. J, Collapsed pollen grain in the same anther as in G. The exine layer, of sporophytic origin, remained wild-type like.
- Figure 7.
Embryo sac development defects in SDH1-1/sdh1-1 mutant plants. Flowers of wild-type and SDH1-1/sdh1-1 plants were emasculated, and whole-mount preparations of ovules were analyzed by DIC microscopy 48 h after emasculation. Embryo sac nuclei are indicated by arrows. A, Ovule from a wild-type Ws plant. The embryo sac is at the terminal developmental stage and contains one secondary nucleus in the central cell, one egg cell nucleus, and two synergid cell nuclei at the micropylar end. B, Ovule harboring a wild-type-like embryo sac in a SDH1-1/sdh1-1 mutant plant. C, Ovule from the same pistil of the ovule shown in B. The embryo sac displays a mutant phenotype, being arrested at the two-nucleate stage. D, Ovule from the same pistil of the ovule shown in B and C, but showing a second mutant phenotype. The two polar nuclei lie side by side but failed to fuse. CC, Central cell nucleus; EC, egg cell nucleus; N, nuclei from an embryo sac arrested at the two-nucleate stage; PN, polar nuclei; SYN, synergid cell nuclei. Bars = 50 μm.
- Figure 8.
Phenotype of SDH1-1 RNAi transgenic plants. A, Schematic diagram of the construct used to down-regulate SDH1-1. The arrows indicate the SDH1-1 sequence (first exon) cloned in both orientations in the RNAi pHELLSGATE4 vector. Sense and antisense SDH1-1 transcribed sequences would be separated by the plant pyruvate orthophosphate dikinase (PDK) intron, generating an ihpRNA. B, Northern-blot analysis of SDH1-1 expression in five RNAi T2 plants and two wild-type controls. Each lane was loaded with 10 μg of total RNA isolated from flowers. The blot was hybridized with a SDH1-1 specific probe and then with an actin probe as loading control. RNAi plants had decreased SDH1-1 mRNA levels. C and D, Pollen abortion in SDH1-1 RNAi plants. Alexander staining was performed on two wild-type and five RNAi T2 anthers. Sections C and D show the staining of one RNAi plant and one wild-type plant, respectively. Quantitative determination of pollen abortion was done by counting viable, purple pollen grains, and aborted, green pollen grains, and the results obtained are shown under the images. Bars = 20 μm. E, Seed set is reduced in SDH1-1 RNAi plants. Seed set per silique was scored for the first eight siliques from five T2 RNAi plants (1–5) and two wild-type control plants (wt). Error bars represent sds.
Additional Files
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figure 1
- Supplemental Data - Supplemental Figure 2
- Supplemental Data - Supplemental Table I
- Supplemental Data - Supplemental Table II
- Supplemental Data - Supplemental Table III
