Iron-sulfur (Fe-S) clusters play an essential role in plants as protein co-factors mediating diverse electron transfer reactions. Because they can react with oxygen to form reactive oxygen species (ROS) and cause cellular damage, the biogenesis of Fe-S clusters is highly regulated. A newly discovered group of 2Fe-2S proteins, termed NEET proteins, was recently proposed to coordinate iron-sulfur, iron, and ROS homeostasis in mammalian cells. Here we report that disrupting the function of AtNEET, the sole member of the NEET protein family in Arabidopsis thaliana, triggers a leaf-associated Fe- and Fe-S-deficiency responses, iron overload in chloroplasts (1.2-1.5 fold), chlorosis, structural damage to chloroplasts, and a high seedling mortality rate. Our findings suggest that disrupting AtNEET function disrupts the transfer of 2Fe-2S clusters from the chloroplastic 2Fe-2S biogenesis pathway to Fe-S proteins, and that uncoupling this process triggers a leaf-associated Fe-deficiency response that results in iron overload in chloroplasts and enhanced ROS accumulation

AtNEET (At5G51720) was amplified by PCR and the H89C sequence was generated by site-directed mutagenesis

For quantitative analysis, protein FASTA sequences for 12 proteins (PetC, PAO, CAO, TIC55, FDC1, Glu1, Glu2, PsaA, PsaB, Nir1, SiR, and SiRB) were imported into Skyline to identify a set of 3-6 unique peptides for each protein based on the TAIR10 proteome. Areas for each peptide signal (or summed areas of multiple peptides) were normalized using the prominent trypsin autolysis peptide (108VATVSLPR115). Peptide digests re-suspended in 25 µL of 5% acetonitrile, 1% formic acid (at 1µg/µL) were separated using a gradient method (50 min). A full-loop injection (10 µL) was loaded onto a C8 trap column (pepmap100, Thermo Scientific). Peptides were eluted from the trap column and separated on a 20 cm x 75 µm inner diameter pulled-needle analytical column packed with HxSIL C18 reversed phase resin (Hamilton Co.) with a step gradient of acetonitrile at 500 nL min-1. The Eksigent Nano 1D plus HPLC system was attached to a Thermo Scientific TSQ Quantiva triple-quadrupole mass spectrometer. LC gradient conditions: Initial conditions were 2% B (A: 0.1% formic acid in water, B: 99.9% acetonitrile, 0.1% formic acid), followed by 2 min ramp to 10% B, gradient 10-40% B over 36 min, ramp to 90% B in 1 min, hold at 90% B for 7 min, ramp back to (1 min) and hold at (5 min) initial conditions. Total run time was 50 min. MRM conditions were: ionization voltage 1600V, Q1 resolution 1.2, Q3 resolution 0.7 (full-width half max), collision gas 3 mTorr