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A leaf is an organ composed of different tissues that fulfill specific functions. We hypothesized that since cells in vascular or mesophyll tissues as well as in stoma are developmentally tuned to operate their functions, mitochondria from these cells could exhibit significant metabolic differences. Using the IMTACT method, mitochondria were isolated from these three specific cell types, and the subsequent proteomes were analyzed. At steady state, mitochondria from vascular and guard cells had a significantly higher abundance of proteins associated with the mtETC, the TCA cycle, and the metabolic use of amino acids (glutamate, proline, isoleucine, leucine, and valine) as alternative substrates. Intriguingly, the mitochondria from guard cells also had a much lower abundance of proteins involved in the translation machinery, thus raising questions about the efficiency of the mitochondrial protein turnover in these cells. In a second step, we carried out the same comparative analysis, but with plants that were subjected to carbon starvation by placing them in prolonged darkness for three or 6 days. For all cell types studied, an increased abundance of proteins involved in branched-chain amino acid metabolism was detected. However, while guard cell mitochondria underwent a drastic reduction in proteins involved in respiration, translation, and RNA editing, suggesting a sharp downregulation of mitochondrial functions, mitochondrial proteomes from mesophyll and vascular cells did not show many differences, except for an increased arginine/proline/glutamate metabolism. Together, the results reported here support a differential regulation of the mitochondrial metabolism among the cell types constituting a leaf, a difference that is exacerbated upon stress.

To propagate their genetic material, flowering plants rely on the production of large amounts of pollen grains that are capable of germinating on a compatible stigma. Pollen germination and pollen tube growth are thought to be extremely energy-demanding processes. This raises the question of whether mitochondria from pollen grains are specifically tuned to support this developmental process. To address this question, we isolated mitochondria from both mature pollen and floral buds using the isolation of mitochondria tagged in specific cell-type (IMTACT) strategy and examined their respective proteomes. Strikingly, mitochondria from mature pollen grains have lost many proteins required for genome maintenance, gene expression, and translation. Conversely, a significant accumulation of proteins associated with the tricarboxylic acid (TCA) cycle, the electron transport chain (ETC), and Ca2+ homeostasis was observed. This supports the current model in which pollen requires large quantities of ATP for tube growth but also identifies an unexpected depletion of the gene expression machinery, aligned with the fact that the mitochondrial genome is actively degraded during pollen maturation. Altogether, our results uncover that mitochondria from mature pollen grains are strategically prepared for action by increasing their respiratory capacity and dismantling their gene expression machinery, which raises new questions about the assembly of respiratory complexes in pollen mitochondria, as they rely on the integration of proteins coded by the nuclear and mitochondrial genomes. In addition, the approach described here opens a new range of possibilities for studying mitochondria during pollen development and in pollen-specific mitochondrial events.

The LYR motif (LYRM)-containing proteins are small eukaryote-specific proteins that have been defined based on the presence of a Lys-Tyr-Arg amino acid motif and a conserved triplet of α-helices. Twelve LYRM proteins were described in humans. They are involved in core mitochondrial processes as subunits or assembly/stabilising factors of mitochondrial complexes. Their function depends on their ability to interact with the acylated form of acyl-carrier proteins (mtACPs), which places these proteins as direct contributors to two intertwined functional processes, energy metabolism and mitochondrial biogenesis. To gain insight into LYRM proteins in Archaeplastida, we first analyzed the Arabidopsis thaliana genome and then a set of organisms representing the different groups of the Archaeplastida clade. This analysis revealed the existence of 17 classes encompassing 10 of the 12 LYRM classes found in humans. Eleven classes exist in Arabidopsis, and six additional classes are present in some organisms but not in Arabidopsis, thus expanding previous observations. Subsequent data mining based on literature, gene expression, and in silico analyses allowed us to speculate about the possible molecular function of some currently uncharacterised LYRMs in plants. Altogether, this study revealed the diversification of the LYRM protein family in Archaeplastida and more globally among eukaryotes, in which the LYRM-mtACP associations represent central molecular systems to regulate mitochondrial biogenesis upon fluctuating growth conditions.

Plastids are complex organelles that vary in size and function depending on the cell type. Accordingly, they can be referred to as amyloplasts, chloroplasts, chromoplasts, etioplasts, or proplasts, to only cite a few. Over the past decades, methods based on density gradients and differential centrifugation have been extensively used for the purification of plastids. However, these methods need large amounts of starting material, and hardly provide a tissue-specific resolution. Here, we applied our IPTACT (Isolation of Plastids TAgged in specific Cell Types) method, which involves the biotinylation of plastids in vivo using one-shot transgenic lines expressing the Translocon of the Outer Membrane 64 (TOC64) gene coupled with a biotin ligase receptor particle and the BirA biotin ligase, to isolate plastids from mesophyll and companion cells of Arabidopsis using tissue specific pCAB3 and pSUC2 promoters, respectively. Subsequently, a proteome profiling was performed, which allowed the identification of 1672 proteins, among which 1342 were predicted to be plastidial, and 705 were fully confirmed according to the SUBA5 database. Interestingly, although 92% of plastidial proteins were equally distributed between the two tissues, we observed an accumulation of proteins associated with jasmonic acid biosynthesis, plastoglobuli (e.g. NAD(P)H dehydrogenase C1, vitamin E deficient 1, plastoglobulin of 34 kDa, ABC1-like kinase 1) and cyclic electron flow in plastids originating from vascular tissue. Besides demonstrating the technical feasibility of isolating plastids in a tissue-specific manner, our work provides strong evidence that plastids from vascular tissue have a higher redox turnover to ensure optimal functioning, notably under high solute strength as encountered in vascular cells.

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I–V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like). This protein was previously found to be part of the bridge domain linking the matrix and membrane arms of the complex I. Phylogenetic analysis suggested that the Arabidopsis (Arabidopsis thaliana) mFDX-like evolved from classical mitochondrial ferredoxins (mFDXs) but lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster, supposedly essential for the electron transfer function of FDXs. Accordingly, our biochemical study showed that AtmFDX-like does not bind an Fe-S cluster and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9-based strategy. These lines did not show any abnormal phenotype under standard growth conditions. However, the characterization of the OXPHOS system demonstrated that mFDX-like is important for the assembly of complex I and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III2 to form supercomplexes.

Plastids are found in all plant cell types. However, most extraction methods to study these organelles are performed at the organ level (e.g., leaf, root, fruit) and do not allow for tissue-specific resolution, which hinders our understanding of their physiology. Therefore, IPTACT (Isolation of Plastids TAgged in specific Cell Types) was developed to isolate plastids in a tissue-specific manner in Arabidopsis thaliana (Arabidopsis). Plastids are biotinylated using one-shot transgenic lines, and tissue specificity is achieved with a suitable promoter as long as such a promoter exists. Cell-specific biotinylated plastids are then isolated with 2.8-µm streptavidin beads. Plastids extracted by IPTACT are suitable for RNA or protein isolation and subsequent tissue-specific OMICs analyses. This method provides the user with a powerful tool to investigate plastidial functions at cell-type resolution. Furthermore, it can easily be combined with studies using diverse genetic backgrounds and/or different developmental or stress conditions.

Membrane-bound organelles are unique features of eukaryotic cell structures. Among them, mitochondria host key metabolic functions and pathways, including the aerobic respiration. In plants, several procedures are available to isolate mitochondria from the other cell compartments, as high-quality purified extracts are often necessary for accurate molecular biology or biochemistry investigations. Protocols based on differential centrifugations and subsequent density gradients are an effective way to extract rather pure and intact mitochondria within a few hours. However, while mitochondria from seedlings, large leaves or tubers are relatively easy to extract, tissue-specific isolation of organelles had remained a challenge. This has recently been circumvented, only in transformable plants though, by the use of affinity-tagged mitochondria and their isolation with magnetic beads. We hereby describe a step-by-step protocol for the rapid and tissue-specific isolation of Arabidopsis thaliana mitochondria, a method named IMTACT (Isolation of Mitochondria TAgged in specific Cell Types). Cell-specific biotinylated mitochondria are isolated with streptavidin magnetic beads in less than 30 min from sampling to final extract. Key steps, enrichment, bead size comparison, and mitochondrial depletion in the sample are also reported in order to facilitate the experimental setup of the user.

In plants, the mitochondrial complex I is the protein complex encompassing the largest number of iron-sulfur (Fe-S) clusters. The whole, membrane-embedded, holo-complex is assembled stepwise from assembly intermediates. The Q and N modules are combined to form a peripheral arm in the matrix, whereas the so-called membrane arm is formed after merging a carbonic anhydrase (CA) module with so-called Pp (proximal) and the Pd (distal) domains. A ferredoxin bridge connects both arms. The eight Fe-S clusters present in the peripheral arm for electron transfer reactions are synthesized via a dedicated protein machinery referred to as the iron-sulfur cluster (ISC) machinery. The de novo assembly occurs on ISCU scaffold proteins from iron, sulfur and electron delivery proteins. In a second step, the preformed Fe-S clusters are transferred, eventually converted and inserted in recipient apo-proteins. Diverse molecular actors, including a chaperone-cochaperone system, assembly factors among which proteins with LYR motifs, and Fe-S cluster carrier/transfer proteins, have been identified as contributors to the second step. This mini-review highlights the recent progresses in our understanding of how specificity is achieved during the delivery of preformed Fe-S clusters to complex I subunits.

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts—likely due to the lack of induction of amino acids (AAs) transport—can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.