Summary: Plant cells contain numerous subcompartments with clearly delineated metabolic functions. Mitochondria represent a very small fraction of the total cell volume and yet are the site of respiration and thus crucial for cells throughout all developmental stages of a plant's life. As such, their isolation from the rest of the cellular components is a basic requirement for numerous biochemical and physiological experiments. Although procedures exist to isolate plant mitochondria from different organs (i.e. leaves, roots, tubers, etc.), they are often tedious and do not provide resolution at the tissue level (i.e. phloem, mesophyll or pollen). Here, we present a novel method called IMTACT (isolation of mitochondria tagged in specific cell types), developed in Arabidopsis thaliana (Arabidopsis) that involves biotinylation of mitochondria in a tissue‐specific manner using transgenic lines expressing a synthetic version of the OM64 (Outer Membrane 64) gene combined with BLRP and the BirA biotin ligase gene. Tissue specificity is achieved with cell‐specific promoters (e.g. CAB3 and SUC2). Labeled mitochondria from crude extracts are retained by magnetic beads, allowing the simple and rapid isolation of highly pure and intact organelles from organs or specific tissues. For example, we could show that the mitochondrial population from mesophyll cells was significantly larger in size than the mitochondrial population isolated from leaf companion cells. To facilitate the applicability of this method in both wild‐type and mutant Arabidopsis plants we generated a set of OM64–BLRP one‐shot constructs with different selection markers and tissue‐specific promoters.

SUMMARY: Iron (Fe) is an essential element for the development and physiology of plants, owing to its presence in numerous proteins involved in central biological processes. Here, we established an exhaustive, manually curated inventory of genes encoding Fe‐containing proteins in Arabidopsis thaliana, and summarized their subcellular localization, spatiotemporal expression and evolutionary age. We have currently identified 1068 genes encoding potential Fe‐containing proteins, including 204 iron‐sulfur (Fe‐S) proteins, 446 haem proteins and 330 non‐Fe‐S/non‐haem Fe proteins (updates of this atlas are available at https://conf.arabidopsis.org/display/COM/Atlas+of+Fe+containing+proteins). A fourth class, containing 88 genes for which iron binding is uncertain, is indexed as ‘unclear’. The proteins are distributed in diverse subcellular compartments with strong differences per category. Interestingly, analysis of the gene age index showed that most genes were acquired early in plant evolutionary history and have progressively gained regulatory elements, to support the complex organ‐specific and development‐specific functions necessitated by the emergence of terrestrial plants. With this gene atlas, we provide a valuable and updateable tool for the research community that supports the characterization of the molecular actors and mechanisms important for Fe metabolism in plants. This will also help in selecting relevant targets for breeding or biotechnological approaches aiming at Fe biofortification in crops.

In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (⁠ $NO3−$⁠) uptake depends mainly on four NRT2 $NO3−$ transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes.

Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]²⁺ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]²⁺ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]²⁺ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]²⁺ clusters, via two-electron reductive coupling of two [2Fe-2S]²⁺ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]²⁺ to [4Fe-4S]²⁺ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.

Iron-containing proteins, including iron-sulfur (Fe-S) proteins, are essential for numerous electron transfer and metabolic reactions. They are present in most subcellular compartments. In plastids, in addition to sustaining the linear and cyclic photosynthetic electron transfer chains, Fe-S proteins participate in carbon, nitrogen, and sulfur assimilation, tetrapyrrole and isoprenoid metabolism, and lipoic acid and thiamine synthesis. The synthesis of Fe-S clusters, their trafficking, and their insertion into chloroplastic proteins necessitate the so-called sulfur mobilization (SUF) protein machinery. In the first part, we describe the molecular mechanisms that allow Fe-S cluster synthesis and insertion into acceptor proteins by the SUF machinery and analyze the occurrence of the SUF components in microalgae, focusing in particular on the green alga Chlamydomonas reinhardtii. In the second part, we describe chloroplastic Fe-S protein-dependent pathways that are specific to Chlamydomonas or for which Chlamydomonas presents specificities compared to terrestrial plants, putting notable emphasis on the contribution of Fe-S proteins to chlorophyll synthesis in the dark and to the fermentative metabolism. The occurrence and evolutionary conservation of these enzymes and pathways have been analyzed in all supergroups of microalgae performing oxygenic photosynthesis.