List of cyanases from plants, fungi and bacteria.

<p>List of cyanases from plants, fungi and bacteria.</p

Coimmunoprecipitation assay demonstrating self-interaction of AtCYN (A) and OsCYN (B).

<p>Although an extremely low amount of OsCYN proteins was detected in the input samples, an identical pattern was shown by OsCYN in the assay. Lane 1: Mock; Lane 2: HA:CYN and FLAG:CYN were detected in the input samples; Lane 3: when anti-HA antibody was added, FLAG:CYN immunoprecipitated with HA:CYN (Lanes 6&8 were controls); Lane 4: when anti-FLAG antibody was added, HA:CYN immunoprecipitated with FLAG:CYN (Lanes 7&9 were controls); Lane 5: Native mouse IgG was used as negative control of antibodies. Bands of HA:CYN and Flag:CYN are indicated with arrows and bands of mouse IgG are indicated with stars.</p

List of primers used in this study.

<p>List of primers used in this study.</p

Gel filtration and cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A).

<p>(A) Gel filtration. High Molecular Weight (HMW) Standard: Thyroglobulin, 669 kDa; Ferritin, 440 kDa; Aldolase, 158 kDa; Conalbumin, 75 kDa and Ovalbumin, 43 kDa. And calculated molecular weight: AtCYN, 210.74 kDa; OsCYN, 211.72 kDa; AtCYN-E94L, 71.64 kDa and AtCYN-S117A, 62.76 kDa. (B) Cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A) at pH 7.7 and 27°C.</p

Decomposition of cyanate by AtCYN and OsCYN <i>in vivo</i>.

<p>Seeds were plated on MS medium containing either 0 mM, 0.5 mM, 1 mM or 2 mM KCNO. Plates were incubated at 4°C for 3 days and then transferred to a growth chamber for 7 days.</p

Alignment of catalytic regions of cyanases from fungus, plant and bacterial species.

<p>Accession numbers for each cyanase are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018300#pone-0018300-t001" target="_blank">Table 1</a>. Residues conserved in all sequences are indicated in white type on a black background, the consensus residue or similar residues in all sequences are indicated in white type on a dark grey background and the consensus residue or similar residues in the sequences of cyanases from Dicotyledoneae and Monocotyledoneae are indicated in black type on a light grey background. The predicted catalytic residues in <i>E. coli</i> are indicated above the alignment.</p

Quantitative RT-PCR analysis of <i>AtCYN</i> transcription.

<p>(A) The <i>AtCYN</i> transcript levels in different plant organs. RL (rosette leaves), CL (cauline leaves); (B) and (C) Samples are the 7-day-old seedlings which grew in the MS medium containing KCNO or NaCl. The <i>AtCYN</i> transcript levels in response to treatment with three KCNO concentrations (B) and in response to treatment with 150 mM NaCl (C); (D) The 7-day-old seedlings from the standard MS medium were transferred to the MS medium containing 1 mM KCNO or 150 mM NaCl, and the samples were harvested at different time points. Error bars represent the standard deviation of three biological replicates.</p

Homology modelling of AtCYN and OsCYN.

<p>(A) The predicted structures of AtCYN (blue) and OsCYN (magentas) were similar to the crystal structure of the EcCYN monomer (green). Ball-and-stick figures represent the conserved catalytic residues Arg96, Glu99 (B) and Ser122 of the EcCYN (C). Red dots indicate chloride ions.</p

Biochemical characteristics of heterologously expressed AtCYN and OsCYN.

<p>The apparent <i>Km</i> and <i>Vmax</i> values were calculated by double-reciprocal plots.</p>a<p>The cyanase reaction was assayed at 27°C, pH 7.7, in the presence of 5 mM KCNO,</p>b<p>The cyanase reaction was assayed at 27°C, pH 7.7, in the presence of 5 mM NaHCO₃.</p

Identification of <i>Atcyn</i> mutant plants and transgenic plants.

<p>(A) Schematic diagram of the <i>AtCYN</i> gene and the T-DNA insertion positions. Gray boxes represent exons. Lines above the gene indicate T-DNA insertion positions. Accession numbers of the <i>Atcyn</i> mutant lines was listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018300#pone-0018300-t004" target="_blank">Table 4</a>. (B) Northern blot analysis of <i>AtCYN</i> transcripts in <i>cyn</i> mutants. Total RNA was isolated from 14-day-old seedlings. Different homozygous individuals identified were analyzed in different lanes. Blot signals (indicated by the arrow) were quantified with ImageJ version 1.4 software and the values are presented below each lane. The 5S rRNA was visualized with ultraviolet light and was the loading control. (C) Quantitative RT-PCR analysis of <i>AtCYN</i> transcripts in Col 0, <i>cyn7</i> and transgenic plants. Error bars represent the standard deviation of three biological replicates. (D) Semi-quantitative analysis of HA:AtCYN and HA:OsCYN in transgenic plants using western blotting (WB). Blot signals (indicated by the arrow) were quantified with ImageJ and the values are presented below the lanes. The large subunit of Rubisco was visualised by Coomassie Brilliant Blue (CBB) and was the total protein loading control (indicated by the arrow).</p