Genome structures and halophyte-specific gene expression of the extremophile Thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and Arabidopsis

Abstract

The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5' untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species.

Figure 1.

Synteny between a T. parvula scaffold and Arabidopsis chromosomes. A Circos plot depicting the T. parvula scaffold 00254 (gray) and Arabidopsis chromosomes 2 (green), 3 (yellow), and 5 (blue) is shown. The predicted T. parvula ORFs were connected to their putative Arabidopsis homologs, with the color of the connecting line designating the Arabidopsis chromosome, to visualize the synteny between two species. The two regions, TP-1 and TP-2, where the genomic sequences were available for three-species comparison are designated with red bands.

Figure 2.

Genome organization for TP-1 (A) and TP-2 (B) in comparison with the organization of orthologous regions in Arabidopsis and T. salsuginea. A, Black arrows in TP-1 show deletions in comparison with AT-1 or TS-1. Red arrows indicate the intergenic space between SOS1 orthologs and their nearest 5′ gene model. B, The inversion break point is indicated by a red arrow. Symbols for the genes are as in Supplemental Tables S1 and S2. Symbols used for transposable elements (TEs) are LTR (retrotransposon with long terminal repeats), SINE (short interspersed transposable elements), LINE (long interspersed transposable elements), MuDR (Mutator-like DNA transposon), Hel (helitron), En-Spm (Enhancer-Suppressor/Mutator transposon), and hAT (hAT family DNA transposon).

Figure 3.

Comparison of the SOS1 omologs in T. parvula, T. salsuginea, and Arabidopsis. The genomic sequence spanning the 5′ intergenic space and SOS1 genes from T. parvula was compared with the corresponding regions in T. salsuginea and Arabidopsis using dot plot (A) and PipMaker plot (B). In B, the PipMaker plots comparing TpSOS1 with TsSOS1 (gray) and AtSOS1 (black) were superimposed to show the differences in similarity. Word size of the PipMaker plot was 7 bp.

Figure 4.

Analysis of SOS1 mRNA abundance in Arabidopsis and T. parvula SOS1. SOS1 mRNA levels were compared between Arabidopsis and T. parvula with RT-PCR (A) and quantitative real-time PCR (B). In A, dilution series of cDNA were used for SOS1 to ensure that the reaction was performed in a linear range. In B, relative mRNA levels were normalized with EF1α as a reference. Fold differences compared with the nonstressed Arabidopsis sample are shown. Error bars indicate the sd from six repeats (two biological and three analytical repeats). Expression patterns of orthologous genes around SOS1 are shown for comparison.

Figure 5.

Comparison of gene expression of translocated genes in T. parvula. Shown are quantitative real-time PCR results comparing the expression of selected orthologous genes from TP-2. Normalization and error bars are as in Figure 4B.

Figure 6.

Frequency of pyrimidine nucleotides in SOS1 5′ UTR sequences of diverse organisms. CT content values (%) are sorted from large to small, and the percentage difference between two consecutive columns is shown above each column. Additional sequences are Chenopodium quinoa (gi:154269387), Salicornia brachiata (gi:214028395), Populus trichocarpa gene POPTR_0010s11130, Manihot esculenta cassava39868.m1, Ricinus communis 29780.t000067, Medicago truncatula Medtr2g043140, Cucumis sativus Cucsa.026500, Arabidopsis lyrata SOS1, Oryza sativa LOC_Os12g44360, Carica papaya evm.TU.supercontig_2.63, Vitis vinifera gene GSVIVT00030673001, Sorghum bicolor Sb08g023290, Zea mays GRMZM2G098494, Brachypodium distachyon Bradi4g00290, Selaginella moellendorfii, Physcomitrella patens, and Chlamydomonas reinhardtii Au9.Cre24.g769600. One hundred nucleotides upstream of the translation start site was taken unless a shorter 5′ UTR was defined for a given species.