Comparison of Arabidopsis BAK1 (AtBAK1) with its homologs in Selaginella.

<p>(A) Functionally important domains, including the leucine-rich repeat and protein kinase domain, are conserved. (B) Phylogenetic analysis of BAK1-like protein sequences. Arrows indicate AtBAK1 and its close homolog Selaginella 85472 (Sm85471). (C) Multiple sequence alignment of AtBAK1 and Sm85471.</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

Percent similarity between amino acid sequences of CYPs.

<p><a href="http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi" target="_blank">www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi</a>). Percent identity between Arabidopsis CYP90s ranged from 32% (between AtCYP90B1 and AtCYP85A1) to 83% (between AtCYP85A1 and AtCYP85A2). A comparison of CYP sequences in Arabidopsis and Selaginella revealed identity values ranging from 31% (between SmCYP90E1 and AtCYP85A1) to 43% (between SmCYP90F1 and AtCYP90A1). SmCYP90E consists of three members that share 77∼86% identity, whereas SmCYP90F has one member, which has 35∼37% identity with the SmCYP90Es. Sequences of known BR biosynthetic enzymes from Arabidopsis were pair-wise compared with select sequences from Selaginella. Percent identity values were calculated using the 2 BLAST program (</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

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

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

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

Anisotropic microstructural evolutions of extruded ZK60 Mg alloy subjected to electropulsing treatment

Electropulsing treatment (EPT) has attracted attentions as an alternative to a conventional furnace heat treatment (FHT). This study investigates the EPT effect on microstructural evolutions in α-Mg alloy. In particular, it clarifies the different kinetics in static recrystallization (SRX) and precipitation depending on the direction of EPT, namely ‘electropulsing anisotropy’, for the first time. EPT process induced an accelerated SRX than FHT in spite of the lower processing temperatures. EPT along the extrusion direction (EPT-ED) exhibited even faster completion of SRX within an hour compared to the process along the transverse direction (EPT-TD). The process also accompanied a faster dissolution of β′2 precipitates. Considering the applied processing temperatures, the athermal effect had a primary contribution to the enhanced microstructural evolutions by the EPT-ED process. EPT-induced charge imbalance improved a dislocation mobility, which increased an atomic diffusive flux. These factors were even intensified in case of EPT-ED than EPT-TD, thereby resulting in the mostly favored SRX and rapid dissolution of precipitates through the former

Enhanced kinetics of microstructural evolution in Ti–6Al–4V through electropulsing treatment

The post-processing of Ti–6Al–4V, which has traditionally been conducted using furnace heat treatment (FHT), is crucial to attaining the optimum microstructure and mechanical performance. This study investigated the microstructural kinetics induced by the electropulsing treatment (EPT) of an extruded Ti–6Al–4V alloy from 50 to 400 s. The results were compared with the conventional microstructural evolution caused by FHT at the same temperature and duration. The remarkable energy efficiency enabled a rapid heating of EPT specimens. EPT enhances the kinetics of several aspects of microstructural evolution, such as phase transformation, static recrystallization, dislocation mobility, and the resultant suppression of grain growth. These phenomena arose from the promoted atomic diffusion due to the combination of the thermal and athermal effects of the electropulses. Grain refinement, amplified solid-solution hardening, and increased phase interfaces improved the microhardness of the EPT specimens. These results demonstrate the potential of EPT to facilitate favorable microstructural modifications in Ti–6Al–4V