The Arabidopsis tetratricopeptide repeat-containing protein TTL1 is required for osmotic stress responses and abscisic acid sensitivity

Abstract

Mutations in the Arabidopsis (Arabidopsis thaliana) TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 1 (TTL1) cause reduced tolerance to NaCl and osmotic stress that is characterized by reduced root elongation, disorganization of the root meristem, and impaired osmotic responses during germination and seedling development. Expression analyses of genes involved in abscisic acid (ABA) biosynthesis and catabolism suggest that TTL1 is not involved in the regulation of ABA levels but is required for ABA-regulated responses. TTL1 regulates the transcript levels of several dehydration-responsive genes, such as the transcription factor DREB2A, and genes encoding dehydration response proteins, such as ERD1 (early response to dehydration 1), ERD3, and COR15a. The TTL1 gene encodes a novel plant protein with tetratricopeptide repeats and a region with homology to thioredoxin proteins. Based on homology searches, there are four TTL members in the Arabidopsis genome with similar intron-exon structure and conserved amino acid domains. Proteins containing tetratricopeptide repeat motifs act as scaffold-forming multiprotein complexes and are emerging as essential elements for plant hormonal responses (such as gibberellin responses and ethylene biosynthesis). In this report, we identify TTL1 as a positive regulator of ABA signaling during germination and seedling development under stress.

Figure 1.

ttl1 alleles shows hypersensitivity to NaCl in root elongation and increased germination rates under osmotic stress and exogenous ABA treatments. A, Photographs of seedlings that were grown on MS agar medium for 1 week and then transferred to MS agar medium for 8 additional days without (left) or with (right) 150 mm NaCl: wild type (C24), stt3a (positive control), and ttl1-1. B, Seedlings of ttl1-2 and ttl1-3 also show NaCl hypersensitivity compared to their respective wild-type control Col-0. Bar = 10 mm. C and D, Root tip morphology of wild-type and ttl1 1-week-old seedlings after treatment with 300 mm mannitol during 72 h (C) and the phenotype observed using Nomarski (D). Bars are 200 and 40 μm, respectively. E, Germination phenotypes of wild-type Col-0 and ttl1-2 seeds sown on paper soaked with liquid MS media supplemented with NaCl (150 mm), KCl (150 mm), mannitol (350 mm), PAC (2 μm), or ABA (2 μm). Plates were placed under long-day photoperiod in a growth chamber for a minimum of 20 d. Two replicates were made for each treatment with similar results (n = 100). Photographs were taken 10 d after sowing.

Figure 2.

Localization of the T-DNA insertions and motif prediction within the At1g53300 locus. A, Exon-intron organization and schematic representation of T-DNA insertions in the At1g53300 gene. White and black boxes correspond to exons and introns, respectively. The white triangle corresponds to the insertion in C24 background and black triangles correspond to insertions in Col-0 background. B, Motif predictions using PROSITE and PFAM in the deduced amino acidic sequence of At1g53300. The light gray and the dark gray correspond to TPR and TRXL motifs, respectively. C, TTL1 transcripts are detected in wild type (Col-0 and C24 backgrounds) and ttl1-4 but not in ttl1-1, ttl1-2, and ttl1-3 seedlings using RT-PCR. The position of the oligonucleotides used for the RT-PCR is indicated with black arrows in A. Pictures show the germination phenotypes of the different alleles in media containing 2 μm ABA. Photographs were taken 10 d after sowing.

Figure 3.

TTL1 belongs to a novel family of proteins composed of four members in the Arabidopsis genome. The phylogram was generated using the PHYLIP algorithm and shows the phylogenetic relationships between TTL1 and closely related proteins from different organisms. TTL1 is grouped in a cluster formed by four Arabidopsis proteins with unknown function, which are closely related with a second cluster formed by four putative orthologous in rice. AGI accession numbers for Arabidopsis sequences and GenBank accession numbers for the rest of the sequences are indicated. Bootstrap values are indicated in each node.

Figure 4.

ttl1 seedlings show root elongation inhibition under osmotic stress. Root elongation of wild type and ttl1 was measured to quantify their sensitivities to several stress agents. Seedlings were grown on MS agar medium for 1 week and then transferred to MS medium with or without various concentrations of NaCl (A), KCl (B), LiCl (C), mannitol (D), Suc (E), or ABA (F). Root elongation (i.e. increase in length after transfer) was determined after 7 d in C24 or after 5 d in Col-0. Error bars indicate ses (n = 15). The experiments were repeated at least three times with similar results. Graphs correspond to one representative experiment.

Figure 5.

ttl1 is specifically affected in ABA responses. Growth and germination responses of wild type (black bars) and ttl1 (white bars) were analyzed for different plant hormones. The hormones analyzed were ABA (A), GA₃ using the GA biosynthesis inhibitor PAC (B), auxin (NAA; C), cytokinin (BA; D), ethylene (using its precursor ACC; E), and BR (F). Inhibition of root and hypocotyl growth is expressed relative to the mean growth without hormones. Error bars indicate ses (n = 15 for root and hypocotyl elongation, n = 100 for germination). Data was collected 5 d after treatments were applied. Two replicates were made for each treatment with similar results and graphs correspond to one representative experiment.

Figure 6.

TTL1 promoter-GUS expression is high in developing tissues, and TTL1 transcript abundance is regulated by ABA and osmotic stress. A, Histochemical localization of GUS activity in transgenic plants carrying the TTL1-GUS-GFP. Five independent T₃ homozygous lines for the construct were grown on MS agar medium for 1 week for seedling staining or grown in soil in a cabinet under long days for adult plant staining. GUS expression was analyzed in three independent plants per line with very similar expression patterns. A to J, Root (5 cm from the apex; A), root (3 cm from the apex; B), elongation zone of the root (1 cm from the apex; C), root tip (D), embryo (E), developing seed (F), silique (G), leaf axil buds (H), silique abscission zone (I), and stem showing GUS staining in guard cells (J). K, Transcript levels of TTL1 analyzed by semiquantitative RT-PCR. Total RNA was isolated from 10-d-old wild-type (Col-0) and ttl1 seedlings treated with water, 100 μm ABA, or 300 mm NaCl for 3 h. β-Tubuline (TUB) expression was used as a control. Two replicates were made for each treatment with similar results.

Figure 7.

ttl1 shows altered expression of several dehydration-responsive genes after ABA treatment. The expression level of several genes encoding enzymes involved in ABA biosynthesis and catabolism (A), ABA signaling (B), and general stress responses (C and D) was analyzed by semiquantitative RT-PCR. Total RNA was isolated from 10-d-old wild-type Col-0 and ttl1 seedlings treated with water or a solution of 100 μm ABA for 3 h. In all experiments, the expression of the constitutive β-tubuline (TUB) gene was used as a control. Two replicates were made for each treatment with similar results.