NEET proteins transfer their 2Fe-2S cluster to apo-Anamorsin.

<p>The 2F-2S cluster transfer reaction was monitored by UV-Vis absorption spectroscopy. The progress of the transfer was plotted versus time. Cluster transfer from NAF-1 (A) and mNT (B) to apo-Anamorsin occurs only when the 2Fe-2S cluster is oxidized and not when reduced with 10 mM sodium dithionite. Replacement of the coordinating His residue with Cys (H114C for NAF-1 and H87C for mNT) inhibits but does not abolish transfer. All traces shown were obtained with 25 μM NAF-1 or mNT (50 μM 2Fe-2S clusters) and 50 μM apo-Anamorsin in 50 mM bis tris, 100 mM NaCl, 2.5 mM DTT, pH 7.0 at 37°C.</p

NEET transfer of 2Fe-2S clusters to apo-Anamorsin is second order.

<p>NAF-1 (A) and mNT (B) transfer to apo-Anamorsin was monitored by UV-Vis absorption spectroscopy for a series of NEET concentrations. For each NEET concentration the ratio 1 NEET dimer per 2 apo-anamorisn was maintained. The rate constant, k_obs, is determined from the fit of the data to an exponential rise and is plotted versus concentration for NAF-1 (C) or mNT (D). The slope of the best line fit was used to determine apparent second order rate constants (<i>k</i>₂) for NAF-1 and mNT, which are 600 ± 90 M^-1 min^-1 and 460 ± 60 M^-1 min^-1 respectively.</p

NEET homodimers can transfer their two 2Fe-2S clusters to Anamorsin.

<p>(A) 2Fe-2S transfer from 50 μM dimeric NAF-1 to 50 μM apo-Anamorsin (two 2Fe-2S clusters per Anamorsin) (shown in red) is compared to 25 μM NAF-1 to 50 μM apo-Anamorsin (one 2Fe-2S cluster per Anamorsin) (shown in blue). Transfer is near complete for the one 2Fe-2S cluster per Anamorsin sample and is approximately 75% complete for the two 2Fe-2S cluster per Anamorsin sample. The latter result shows that at least half of the Anamorsin is capable of receiving two 2Fe-2S clusters from a single NAF-1 homodimer. (B) Anamorsin following a transfer reaction with NAF-1 was purified and analyzed by ESI-MS (shown in red). Mass spectra of apo-Anamorsin (solid black line) is compared to the major peak for post-transfer Anamorsin (solid red line) which shows an increase in the mass corresponding to the incorporation of two 2Fe-2S clusters. There is also a minor peak at 35856 Da that likely corresponds to Anamorsin with a single 2Fe-2S cluster and an iron ion bound that may be a remnant of a cluster that degraded during the sample preparation process. <i>E</i>. <i>coli</i>-purified Anamorsin is shown for comparison (dotted line) which shows Anamorsin containing a single 2Fe-2S cluster.</p

Biolayer interferiometry shows a direct protein-protein interaction of NAF-1 and mNT with apo-Anamorsin.

<p>Sensorgrams for the binding of holo-NAF-1 (A) and holo-mNT (B) to biotinylated apo-Anamorsin immobilized to streptavidin-coated biosensors are shown. The association was followed for 900 seconds (rising signal) followed by 1800 seconds of dissociation (decaying signal). The data was fit to a one-to-one model (black curves). On- and off-rates were determined from the fits for each NEET-Anamorsin concentration (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139699#pone.0139699.t002" target="_blank">Table 2</a>).</p

NEET-apo-Anamorsin association and dissociation rates determined by biolayer interferometry.

<p>NEET-apo-Anamorsin association and dissociation rates determined by biolayer interferometry.</p

Structures and UV-Vis absorption spectra of NEET proteins and Anamorsin.

<p>A. (Top) Crystal structures of mNT (PDB code: 2QH7,), NAF-1 (PDB code: 3FNV); (Middle) NEET 2Fe-2S cluster with 3-Cys:1His coordination; (Bottom) Absorption spectra of 25 μM mNT and NAF-1. B. (Top) Crystal structure of the N-terminal domain of Anamorsin (PDB code: 2YUI) with an added schematic of the unstructured 2Fe-2S cluster binding domain; (Middle) Representative Anamorsin 2Fe-2S cluster with 4-Cys coordination (from ferredoxin, PDB code: 1RFK); (Bottom) Absorption spectrum of 43 μM Anamorsin isolated from <i>E</i>. <i>coli</i>.</p

NEET proteins provide a link between the ISC and CIA pathways.

<p>mNT on the OMM and NAF-1 on the MAM receive 2Fe-2S clusters produced inside the mitochondria by the ISC system. Both mNT and NAF-1 transfer these 2Fe-2S clusters to Anamorsin. Anamorsin receives electrons from the diflavin reductase NDOR1 and supplies them to the CIA system as an early step necessary for the production of 4Fe-4S clusters. This step requires the holo form of Anamorsin. Both mNT and NAF-1 can provide parallel routes linking CIA to ISC. CIA produced clusters are targeted to proteins in the cytosol and the nucleus and are important for cell metabolism, maintenance and proliferation.</p

NEET proteins can transfer their clusters to either of Anamorsin 2Fe-2S cluster-binding sites.

<p>Anamorsin mutants containing a single cluster-binding site are shown; the other cluster site was disrupted by replacing all of the Cys residues with Ser. (A) Schematics of Anamorsin-C1 (green) and Anamorsin-C2 (magenta) are shown. 2Fe-2S cluster transfer from NAF-1 (B) or mNT (C) to each single cluster binding apo-Anamorsin mutant was monitored by UV-Vis absorption spectroscopy. Traces were obtained with 25 μM NAF-1 or mNT (50 μM 2Fe-2S clusters) and 50 μM apo-Anamorsin.</p

Table_4_Multi-omic characterization of bifunctional peroxidase 4-coumarate 3-hydroxylase knockdown in Brachypodium distachyon provides insights into lignin modification-associated pleiotropic effects.xlsx

A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon. However, like many other lignin-modified plants, C3H/APX-KDs show unpredictable pleiotropic phenotypes, including stunted growth, delayed senescence, and reduced seed yield. A system-wide level understanding of altered biological processes in lignin-modified plants can help pinpoint the lignin-modification associated growth defects to benefit future studies aiming to negate the yield penalty. Here, a multi-omic approach was used to characterize molecular changes resulting from C3H/APX-KD associated lignin modification and negative growth phenotype in Brachypodium distachyon. Our findings demonstrate that C3H/APX knockdown in Brachypodium stems substantially alters the abundance of enzymes implicated in the phenylpropanoid biosynthetic pathway and disrupt cellular redox homeostasis. Moreover, it elicits plant defense responses associated with intracellular kinases and phytohormone-based signaling to facilitate growth-defense trade-offs. A deeper understanding along with potential targets to mitigate the pleiotropic phenotypes identified in this study could aid to increase the economic feasibility of lignocellulosic biofuel production.</p

Table_3_Multi-omic characterization of bifunctional peroxidase 4-coumarate 3-hydroxylase knockdown in Brachypodium distachyon provides insights into lignin modification-associated pleiotropic effects.xlsx

A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon. However, like many other lignin-modified plants, C3H/APX-KDs show unpredictable pleiotropic phenotypes, including stunted growth, delayed senescence, and reduced seed yield. A system-wide level understanding of altered biological processes in lignin-modified plants can help pinpoint the lignin-modification associated growth defects to benefit future studies aiming to negate the yield penalty. Here, a multi-omic approach was used to characterize molecular changes resulting from C3H/APX-KD associated lignin modification and negative growth phenotype in Brachypodium distachyon. Our findings demonstrate that C3H/APX knockdown in Brachypodium stems substantially alters the abundance of enzymes implicated in the phenylpropanoid biosynthetic pathway and disrupt cellular redox homeostasis. Moreover, it elicits plant defense responses associated with intracellular kinases and phytohormone-based signaling to facilitate growth-defense trade-offs. A deeper understanding along with potential targets to mitigate the pleiotropic phenotypes identified in this study could aid to increase the economic feasibility of lignocellulosic biofuel production.</p