The Arabidopsis TETRATRICOPEPTIDE THIOREDOXIN-LIKE Gene Family Is Required for Osmotic Stress Tolerance and Male Sporogenesis

Phylogenetic relationship among individual TPR motifs. An unrooted tree was built using the six TPR sequences of the four Arabidopsis proteins and TPR sequences from TTL orthologs from phylogenetically distant species such moss, lycophytes, and monocots. [See online article for color version of this figure.]

Phylogenetic and sequence analyses of TRXL domains. A, Phylogenetic analysis of the TRXL domain of the TTLs from Arabidopsis, rice, P. patens, and S. moellendorffii. In the analysis, the protein sequence of At3g17880, a genuine thioredoxin, was included. B, Detail of the alignment of the TRXL domains, including the two-Cys motif (WCGPC) required for thioreductase activity. As shown, homologous regions from the TRXL domains of TTLs lack at least one of the two Cys residues that are essential for reductase activity. [See online article for color version of this figure.]

Molecular characterization of ttl mutants. A, ttl1, ttl2, ttl3, and ttl4 mutant alleles. The corresponding T-DNA line from SALK is indicated at the top. Gray boxes represent exons, and lines represent introns. ATG and stop codons are indicated. White boxes indicate the 5′ and 3′ untranslated regions. T-DNA insertion sites (not drawn to scale) are represented by triangles. The primers used for PCR genotyping are indicated by arrows (see “Materials and Methods” for primer sequences). LB, Left border. B, Expression analysis of the four TTL genes in the wild type (WT) and ttl mutants by RT-PCR. RNA was extracted from flowers, which is the only tissue where TTL2 expression was detected. The gene-specific primers designed to amplify cDNA fragments are detailed in “Materials and Methods.” The tubulin gene (Tub) was used as a positive control for the RT-PCR.

Root growth responses of ttl mutants to mannitol stress. Root elongation of single, double, and triple ttl mutants was measured, and root growth is expressed as the percentage relative to wild-type seedlings grown in the same conditions. Results are means of three independent experiments ± sd. Asterisks indicate significant differences between samples as determined by t test (* P < 0.1, ** P < 0.05).

TTL4 loss of function increases tolerance to mild NaCl stress. A, Relative fresh weight of single ttl mutants and selected double and triple ttl mutants in MS medium or MS medium supplemented with 120 mm NaCl. All genotypes containing the ttl4 mutation displayed improved growth in medium supplemented with NaCl. Asterisks indicate significant differences between samples as determined by t test (* P < 0.05). B, Images of seedlings grown on MS agar medium for 1 week and transferred to MS agar medium for an additional 7 d without (left) or with (right) 120 mm NaCl. C, Sodium content in the shoot of the seedlings depicted in B. The asterisk indicates a significant difference between samples as determined by t test (* P < 0.05). WT, Wild type. [See online article for color version of this figure.]

Tissue-specific expression pattern of TTL genes by promoter-reporter (GUS) fusions. GUS histological staining in transgenic Arabidopsis lines containing pTTL::GUS constructs is shown. The samples depicted are 2-d-old seedlings (A–D), 10-d-old seedlings (E–H), and flowers (I–M).

TTL genes show differential expression responses to NaCl and mannitol stresses. Transgenic lines harboring TTL promoter::GUS fusions were grown for 4 d and then transferred to control medium or medium supplemented with 120 mm NaCl or 300 mm mannitol for 24 h before staining for GUS activity. The images show whole seedlings, roots, and cotyledons.

TTL genes are required for male gametophytic development. Pollen and siliques from wild-type (WT) and ttl1/ttl2/ttl3/TTL4/ttl4 plants were analyzed by light microscopy. ttl1/ttl2/ttl3/TTL4/ttl4 plants showed abnormal, shrunken pollen grains (indicated by arrowheads). ttl1/ttl2/ttl3/TTL4/ttl4 plants do not support normal seed development, showing approximately 50% aborted seeds. [See online article for color version of this figure.]