Expression analysis of recombinant AtSINAL7.

<p>The <i>E. coli</i> cell extracts electrophoresed on SDS-PAGE were revealed with Coomassie brilliant blue staining. Lane 1: Protein extract from uninduced bacteria. Lane 2: Soluble proteins obtained after 5 h IPTG induced bacterial culture. Lane 3: Purified AtSINAL7 fraction stained with Coomassie blue. Lane 4: Western blott analysis of the purified AtSINAL7 fraction revealed using anti-His antobodies. MW (kDa), PageRuler Prestained Protein Ladder (Fermentas).</p

Far-UV CD Spectra of recombinant AtSINAL7 and AtSINAL7 mutants.

<p>Far-UV CD spectra were obtained using a Jasco J-810 spectropolarimeter (Jasco International Co.) over the wavelength range from 190 to 250 nm, at 25°C. Measurements were performed in a 0.2 cm quartz cuvette at a rate of 100 nm.min^-1, bandwidth of 1 nm, response time of 2 s, data pitch of 1 nm, and accumulation of 10. CD data are shown as the mean residue ellipticity (deg.cm2.dmol^-1) obtained after subtracting the baseline, smoothing, and data normalization. CD spectra for AtSINAL7 (solid line), AtSINAL7K23A (dotted line), AtSINAL7K214A (dashed line) and AtSINAL7K23AK124A (dashed and dotted line) were recorded in 20 mM Na-phosphate buffer, pH 7.4.</p

E3 ligase activity of AtSINAL7 mutants.

<p><i>In vitro</i> self-ubiquitination reactions were performed incubating purified His-SL7K23A (lane 2), His-SL7K124A (lane 4), His-SL7K23AK124A (lane 6) and His-AtSINAL7 (lane 7) in the presence of cMyc-ubiquitin, yeast E1, E2 UbcH2 as described in the Methods section. Reaction products were separated on 15% (w/v) SDS-PAGE and ubiquitinated proteins were detected by immunoblot analysis using anti-cMyc antibody. MW (kDa), PageRuler Prestained Protein Ladder (Fermentas).</p

Analysis of AtSINAL7 mRNA levels (A) AtSINAL7 transcript expression profile in different organs from <i>A. thaliana</i>.

<p>AtSINAL7 transcript levels relative to CBP control gene expression were determined by RT-qPCR in several <i>A. thaliana</i> organs. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). <b>(B) Time-course expression of AtSINAL7 transcripts.</b> Determination of the transcription levels of <i>AtSINAL7</i> at 0, 4, 8, 12, 16, 20, and 24 hours under long day growth conditions. Transcripts levels are plotted as relative values using the cap binding protein (At5g44200) mRNA as an internal control. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). <b>(C) Effect of the UV–B treatment on the AtSINAL7 transcript levels in wild type <i>A. thaliana</i> inflorescences.</b> The <i>AtSINAL7</i> transcript expression in <i>A. thaliana</i> inflorescences (stage 12) relative to <i>cpk3</i> (calcium-dependent protein kinase 3, At4g23650) was determined after 4 h of UV–B exposure. Control plants were protected from UV–B irradiation using polyester filters (see Methods). Data shown represent at least three independent experiments (significant statistical differences determined using t test, P <0.05).</p

AtSINAL7 E3 ligase activity and identification of self-ubiquitinated AtSINAL7.

<p><b>(A) E3 ligase activity of AtSINAL7. </b><i>In vitro</i> self-ubiquitination reactions were performed using cMyc-ubiquitin as a substrate as described in the Methods section. Lane 1: Ubiquitination buffer. Lane 2: Ubiquitination buffer + E1. Lane 3: Ubiquitination buffer + E2. Lane 4: Ubiquitination buffer + cMyc-ubiquitin. Lane 5: Ubiquitination buffer + AtSINAL7. Lane 6: Ubiquitination buffer + E1 + E2. Lane 7: Ubiquitination buffer + E1 + E2 + cMyc-ubiquitin. Lane 8: Ubiquitination buffer + E1 + E2 + cMyc-ubiquitin + AtSINAL7. AtSINAL7/ubiquitin conjugates were resolved by electrophoresis on 15% (w/v) SDS-PAGE and detected by immunoblot analysis using anti-cMyc antibody. <b>(B) Identification of self-ubiquitinated AtSINAL7.</b> AtSINAL7/ubiquitin conjugates were electrophoresed on 15% (w/v) SDS-PAGE and detected by immunoblot analysis using anti-HIS (Qiagen) and polyclonal anti-AtSINAL7. Lane 1: Control containing the ubiquitination assay buffer alone. Lane 2 and 3: purified His-AtSINAL7 incubated in the complete ubiquitination reaction medium containing the cMyc-ubiquitin substrate and the yeast E1 and UbcH2 E2 components. Lane 4: purified His-AtSINAL7 alone. MW (kDa), PageRuler Prestained Protein Ladder (Fermentas).</p

Predicted subcellular localization of AtFH (At4g03240).

<p>^a TargetP 1.1 Server (<a href="http://www.cbs.dtu.dk/services/TargetP" target="_blank">http://www.cbs.dtu.dk/services/TargetP</a>)</p><p>^b MitoProt II–v1.101 (<a href="http://ihg.gsf.de/ihg/mitoprot.html" target="_blank">http://ihg.gsf.de/ihg/mitoprot.html</a>)</p><p>^c ChloroP 1.1 Server (<a href="http://www.cbs.dtu.dk/services/ChloroP/" target="_blank">http://www.cbs.dtu.dk/services/ChloroP/</a>)</p><p>^d GTP—Green Targeting Predictor & ATP2—Ambiguous Targeting Predictor 2 (<a href="http://www.plantco.de/gtp" target="_blank">http://www.plantco.de/gtp</a>).</p><p>Predicted subcellular localization of AtFH (At4g03240).</p

Subcellular localization of AtFH.

<p>The plasmid pZP212 containing the coding sequence of AtFH-GFP was introduced into Arabidopsis thaliana Col-0 plants by the floral dip method. Protoplasts were isolated from the resulting transgenic plants and analyzed by confocal microscopy. (A) Mitotracker staining showing mitochondria (excitation 543 nm/emission 576 nm); (B) GFP fluorescence (excitation 488 nm/emission 510 nm); (C) Chlorophyll autofluorescence (excitation 488 nm/emission 650 nm); (D) Overlay of A and B showing coincidence of GFP localization and Mitotracker (yellow); (E) Overlay of B and C showing the coincidence of GFP localization and chlorophyll autofluorescence (yellow); (F) Phase contrast image of the protoplast analyzed.</p

Photosynthetic parameters in <i>as-AtFH</i> and wt leaves.

<p>Fv/Fm, Maximum quantum yield of PSII; ϕ PSII, Quantum yield of PSII; qP, Photochemical quenching; NPQ, Non-photochemical quenching. Values are the mean ± standard deviation of at least three leaves from 10 individual plants.</p><p>Asterisks (*) indicate statistically different results (P < 0.05).</p><p>Photosynthetic parameters in <i>as-AtFH</i> and wt leaves.</p

Analysis of the presence of AtFH in isolated chloroplasts and mitochondria from <i>A</i>. <i>thaliana</i> by western blot in wt and <i>as-AtFH-1</i> plants.

<p>AtFH was detected using anti-AtFH antibodies. Anti-ADPGlc PPase and anti-NAD9 antibodies were used as controls to asses the purity of chloroplasts and mitochondria, respectively.</p

Quantification of chlorophyll and iron contents.

<p>(A) Quantification of chlorophyll a, b and total chlorophyll in wt (black bars), <i>as-AtFH-1</i> (grey bars), and <i>as-AtFH-2</i> (white bars) plants. (B) Analysis of total iron content in wt and <i>as-AtFH</i> plants. Asterisks indicate a statistically different result from the control value (P < 0.05). Values are the mean ± standard deviation of at least four independent replicates.</p