No accessions display differential growth of roots during Copper treatment.

<p>Col-0, Limeport CS8070, Limeport CS28464, Santa Clara CS8069, Santa Clara CS28722, Berkeley CS28067 and Berkeley CS8068 were germinated and grown on solidified one-half Murashige and Skoog supplemented with the indicated concentrations of CuCl₂. After 15 days primary root length was measured for each treatment. Data represent the mean (N = 5; ± SE). Comparisons between accessions were done by ANOVA using Tukey (P<0.05).</p

No accessions display differential growth of roots during Nickel treatment.

<p>Col-0, Limeport CS8070, Limeport CS28464, Santa Clara CS8069, Santa Clara CS28722, Berkeley CS28067 and Berkeley CS8068 were germinated and grown on solidified one-half Murashige and Skoog supplemented with Ni(NO₃)₂ at the indicated concentrations. After 15 days primary root length was measured for each treatment. Data represent the mean (N = 5; ± SE). Comparisons between accessions were done by ANOVA using Tukey (P<0.05).</p

Locations of <i>A</i>. <i>thaliana</i> collection and serpentine soil presence in California.

<p>Each dot on the map represents a different locality. The arrow indicates the only accession within 400 M of known serpentine soils.</p

Comparison of high quality SNPs between accessions.

<p>Comparison of high quality SNPs between accessions.</p

Comparison of Arabidopsis BRI1 (AtBRI1) with its closest homolog from Selaginella.

<p>(A) Multiple sequence alignment between AtBRI1 and the Selaginella sequence Sm99902 was performed using ClustalW. Underlining delimits the domains. Red triangles indicate residues identified as being functionally important by mutation studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081938#pone.0081938-Vert1" target="_blank">[41]</a> and blue triangles are key residues that participate in BL binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081938#pone.0081938-She1" target="_blank">[42]</a>. (B) Schematic representation of the domains identified in AtBRI1. A key for the colors and shapes used is provided below the diagram. Scale bar = 100 bp. (C) Phylogenetic tree of AtBRI1 and closely related sequences from maize, rice, Physcomitrella, and Selaginella. Arrows indicate AtBRI1 and Sm99902. Scale bar = genetic distance of 200.</p

Predicted subcellular localization of CYPs and their putative reductases.

<p>(A) Multiple sequence alignment of CYP amino acid sequences from Selaginella and Arabidopsis. Sequences corresponding to functional domains identified in AtCYP90B1 are underlined. Box shading was carried out using BOXSHADE 3.21. Dashes represent gaps. Letters in black and gray backgrounds indicate 100% and 50% conservation among the sequences, respectively. The N-terminal regions up to the first 124 amino acids are shown. (B) Prediction of the subcellular localization of the NADPH-cytochrome P450 reductases (CPRs). The PSORT program was used to predict and score the calculation. (C) Phylogenetic tree of CPRs from Arabidopsis, maize, rice, Physcomitrella, and Selaginella. CPRs from the same species or from <i>Zea mays</i> (Zm) and <i>Oryza sativa</i> (Os) combined are bracketed on the right. The <i>Chlamydomonas reinhardtii</i> CPR sequence was used as an outgroup. The broken line for the outgroup indicates a genetic distance of 400. Scale bar = genetic distance of 200.</p

No accessions display differential growth of roots during Zinc treatment.

<p>Col-0, Limeport CS8070, Limeport CS28464, Santa Clara CS8069, Santa Clara CS28722, Berkeley CS28067 and Berkeley CS8068 were germinated and grown on solidified one-half Murashige and Skoog supplemented with Zn(NO₃)₂ at the indicated concentrations. After 15 days primary root length was measured for each treatment. Data represent the mean (N = 5; ± SE). Comparisons between accessions were done by ANOVA using Tukey (P<0.05).</p

Identification of BR signaling components by comparative sequence analysis.

<p>’β (SERINE/THREONINE PROTEIN PHOSPHATASE 2A B’β). Percent identity values were obtained after performing a BLASTX search of the Phytozome database. The number of Arabidopsis gene copies is given in parentheses after each locus name. Abbreviations: BRI1 (BRASSINOSTEROID INSENSITIVE1), BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE1), BKI1 (BRI1 KINASE INHIBITOR1), BSK1 (BR-SIGNALING KINASE1), CDG1 (CONSTITUTIVE DIFFERENTIAL GROWTH1), BIN2 (BRASSINOSTEROID-INSENSITIVE 2), BSU1 (BRI1 SUPPRESSOR1), BZR1 (BRASSINAZOLE-RESISTANT1), BES1 (BRI1-EMS-SUPPRESSOR1), BSK1 (BR-SIGNALING KINASE1), and PP2A B</p

Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

<div><p>Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte <i>Selaginella moellendorffii</i>. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and <i>epi</i>-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.</p></div

Response of Selaginella to <i>epi-</i>brassinolide (<i>epi-</i>BL) and propiconazole (Pcz).

<p>(A) Morphologies after treatment with mock, 10⁻⁵M <i>epi-</i>BL, and 10⁻⁵M Pcz for two weeks. Scale bar = 0.5 cm. (B) Average shoot lengths after treatment. Letters above each bar indicate significant differences compared with the mock treatment. Statistical significance was determined using one-way ANOVA (P<0.01, n = 7). Error bars are standard deviations. (C) Average root lengths after treatment. Letters above each bar indicate significant differences compared with the mock treatment. Statistical significance was determined using student t-test (P<0.01, n≥3). Error bars are standard deviations.</p