Article Figures & Data
Figures
- Figure 1.
Symbiotic Phenotypes of sst1-1 and sst1-2.
Plants were inoculated with M. loti and grown in quartz sand without mineral nitrogen for 4 weeks in a greenhouse, as described in Methods.
(A) Wild type, left; sst1-1, middle; sst1-2, right. Bar = 2 cm.
(B) to (D) Close-up views of nodules from the wild type (B), sst1-1
(C), and sst1-2 (D). Bars = 1 mm.
- Figure 2.
Comparison of Wild-Type and sst1 Mutant Nodule Ultrastructure at 21 d after Inoculation.
Plants were inoculated with M. loti and grown in clay beads without mineral nitrogen in a greenhouse, as described in Methods.
(A) Light micrograph of a mature wild-type nodule showing infected cells packed with bacteria.
(B) Light micrograph of an sst1-1 mutant nodule showing infected cells containing several vacuoles (arrows), which may be associated with lysis of the bacteroids.
(C) Transmission electron micrograph of an infected cell of a mature wild-type nodule containing bacteroids within intact symbiosomes surrounded by SM (arrow).
(D) Transmission electron micrograph of an infected cell of a mature sst1-1 mutant nodule showing the formation of a lytic vacuole (asterisk).
(E) Transmission electron micrograph showing strong immunogold labeling of NifH protein inside bacteroids (b) of a wild-type nodule.
(F) Transmission electron micrograph showing weak immunogold labeling of NifH protein inside bacteroids (b) of an sst1-1 mutant nodule.
(G) Transmission electron micrograph showing immunogold labeling of leghemoglobin in the cell cytoplasm (c) of a wild-type nodule.
(H) Immunogold labeling of leghemoglobin in the cell cytoplasm (c) of an sst1-1 mutant nodule showing slightly reduced levels of the protein.
Bars = 50 μm in (A) and (B), 1 μm in (C) and (D), and 500 nm in (E) to (H).
- Figure 3.
Growth, Nodulation, and Nitrogen Fixation Phenotypes of sst1-2.
Plants were inoculated with M. loti and grown in quartz sand without mineral nitrogen in a greenhouse, as described in Methods. Plant fresh weight, number of nodules, nodule fresh weight, and acetylene reduction activity (ARA) in the wild-type Gifu and the sst1-2 mutant during plant development are shown. All values are means of three determinations, and the vertical bars indicate se.
- Figure 4.
Map-Based Cloning of Sst1 and Molecular Analysis of the Mutations.
(A) Genetic map of the Sst1 region with markers and numbers of recombinants indicated above the line. Genetic distance in centimorgan (cM) is indicated below the line. The physical map of the sequenced TAC clone LjT17O14a is shown. The Sst1 gene is indicated as a black arrow, and a duplicated region containing exon 8 to exon 12 of Sst1 is indicated as a white arrow. Retrotransposon-related sequences are indicated as gray boxes.
(B) Intron–exon structure of the Sst1 gene with sizes (bp) of exons and introns indicated above and below the line, respectively. Black arrowheads mark the positions of mutations in the sst1-1 and sst1-2 alleles.
(C) PCR analysis of Sst1 genomic DNA (lanes 2 and 3) and cDNA (lanes 4 and 5) from wild-type plants (lanes 2 and 4) and sst1-2 plants (lanes 3 and 5) using primers flanking the sst1-2 mutation site. Note the absence of a PCR amplicon from cDNA of the mutant (lane 5).
(D) PCR performed on cDNA from the wild type (lane 2) and sst1-1 (lane 3) using primers flanking the sst1-1 mutation site. PCR amplicons were digested with BglII before gel electrophoresis, which resulted in two fragments of 217 and 170 bp in the wild type. Note the downshift in the smaller of these bands from sst1-1, corresponding to a loss of nine nucleotides in the mutant transcript.
(E) Indication of the mutations in the sst1-1 and sst1-2 alleles. Light gray arrowheads indicate the splice sites in the wild type (WT), whereas the black arrowhead indicates the position of the alternative splice site of the sst1-1 mutant allele. The nucleotide changes in the two mutant alleles are indicated in boldface.
- Figure 5.
Features of the SST1 Amino Acid Sequence.
Putative transmembrane domains are underlined. The three amino acids, RVM, deleted in the sst1-1mutant protein are indicated in boldface. Conserved residues of the STAS domain are highlighted in light gray.
- Figure 6.
Developmental Regulation of Sst1 Expression.
Plants were inoculated with M. loti and grown under sterile conditions on agar plates, as described in Methods.
(A) Relative transcript level for Sst1 in different organs.
(B) Time course of Sst1 expression in inoculated roots, including nodules (black bars). Transcript levels for leghemoglobin (gray bars) are included for comparison. Note the logarithmic scale on the y axis. All transcript levels are expressed relative to those of ubiquitin in the same sample.
- Figure 7.
Phylogenetic Analysis of Sulfate Transporters from Arabidopsis thaliana and LjSST1 from Lotus japonicus.
The nomenclature of Hawkesford (2003) was used: AtSulftr1;1 = At4g08620, AtSulftr1;2 = At1g78000, AtSulftr1;3 = At1g22150, AtSulftr2;1 = At5g10180, AtSulftr2;2 = At1g77990, AtSulftr3;1 = At3g51895, AtSulftr3;2 = At4g02700, AtSulftr3;3 = At1g23090, AtSulftr3;4 = At3g15990, AtSulftr3;5 = At5g19600, AtSulftr4;1 = At5g13550, AtSulftr4;2 = At3g12520, AtSulftr5;1 = At1g80310, and AtSulftr5;2 = At2g25680. Full-length amino acid sequences were aligned using ClustalW (http://clustalw.genome.ad.jp/) and analyzed with the PHYLIP software package (http://www.csc.fi/molbio/progs/phylip/doc/main.html). Bootstrap values were obtained from 100 replicates. The branch lengths indicate the frequency of the corresponding clade in the set of bootstrap trees.
- Figure 8.
Functional Complementation of Yeast Sulfate Transporter Mutant CP154-7A by SST1.
Yeast strains were grown on minimal medium containing 0.5 mM MgSO4 as sole sulfur source. Cells were spotted in a 10-fold dilution series from left to right. A wild-type strain transformed with the expression vector p112A1NE (top row) was included as a positive control for mutant strain CP154-7A transformed with Sst1 cDNA in p112A1NE (middle row). The mutant strain containing p112A1NE (bottom row) was included as a negative control.
- Figure 9.
Proposed Role of SST1 in Nodule Symbiosome Sulfate Transport.
Based on data presented here and elsewhere (Wienkoop and Saalbach, 2003), SST1 is proposed to transport sulfate across the SM from the plant cell cytoplasm into the symbiosome space (SS), where it is available to nitrogen fixing bacteroids (B). Anion transport into symbiosomes is favored by the net positive charge on the inside surface of the SM, which is generated by an H+-ATPase (data not shown).
