| 1. |
- Boussardon, Clément, et al.
(författare)
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Cell Type–Specific Isolation of Mitochondria in Arabidopsis
- 2022. - 1
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Ingår i: Plant Mitochondria. - New York : Humana Press. - 9781071616529 - 9781071616536 - 9781071616550 ; , s. 13-23
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Bokkapitel (refereegranskat)abstract
- 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.
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| 2. |
- Boussardon, Clément, et al.
(författare)
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Comparison of plastid proteomes points towards a higher plastidial redox turnover in vascular tissues than in mesophyll cells
- 2023
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 74:14, s. 4110-4124
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Boussardon, Clément, et al.
(författare)
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Novel Cytonuclear Combinations Modify Arabidopsis thaliana Seed Physiology and Vigor
- 2019
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Ingår i: Frontiers in Plant Science. - : Frontiers Media S.A.. - 1664-462X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Dormancy and germination vigor are complex traits of primary importance for adaptation and agriculture. Intraspecific variation in cytoplasmic genomes and cytonuclear interactions were previously reported to affect germination in Arabidopsis using novel cytonuclear combinations that disrupt co-adaptation between natural variants of nuclear and cytoplasmic genomes. However, specific aspects of dormancy and germination vigor were not thoroughly explored, nor the parental contributions to the genetic effects. Here, we specifically assessed dormancy, germination performance and longevity of seeds from Arabidopsis plants with natural and new genomic compositions. All three traits were modified by cytonuclear reshuffling. Both depth and release rate of dormancy could be modified by a changing of cytoplasm. Significant changes on dormancy and germination performance due to specific cytonuclear interacting combinations mainly occurred in opposite directions, consistent with the idea that a single physiological consequence of the new genetic combination affected both traits oppositely. However, this was not always the case. Interestingly, the ability of parental accessions to contribute to significant cytonuclear interactions modifying the germination phenotype was different depending on whether they provided the nuclear or cytoplasmic genetic compartment. The observed deleterious effects of novel cytonuclear combinations (in comparison with the nuclear parent) were consistent with a contribution of cytonuclear interactions to germination adaptive phenotypes. More surprisingly, we also observed favorable effects of novel cytonuclear combinations, suggesting suboptimal genetic combinations exist in natural populations for these traits. Reduced sensitivity to exogenous ABA and faster endogenous ABA decay during germination were observed in a novel cytonuclear combination that also exhibited enhanced longevity and better germination performance, compared to its natural nuclear parent. Taken together, our results strongly support that cytoplasmic genomes represent an additional resource of natural variation for breeding seed vigor traits.
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| 4. |
- Boussardon, Clément, et al.
(författare)
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The RPN12a proteasome subunit is essential for the multiple hormonal homeostasis controlling the progression of leaf senescence
- 2022
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Ingår i: Communications Biology. - : Nature Publishing Group. - 2399-3642. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- The 26S proteasome is a conserved multi-subunit machinery in eukaryotes. It selectively degrades ubiquitinated proteins, which in turn provides an efficient molecular mechanism to regulate numerous cellular functions and developmental processes. Here, we studied a new loss-of-function allele of RPN12a, a plant ortholog of the yeast and human structural component of the 19S proteasome RPN12. Combining a set of biochemical and molecular approaches, we confirmed that a rpn12a knock-out had exacerbated 20S and impaired 26S activities. The altered proteasomal activity led to a pleiotropic phenotype affecting both the vegetative growth and reproductive phase of the plant, including a striking repression of leaf senescence associate cell-death. Further investigation demonstrated that RPN12a is involved in the regulation of several conjugates associated with the auxin, cytokinin, ethylene and jasmonic acid homeostasis. Such enhanced aptitude of plant cells for survival in rpn12a contrasts with reports on animals, where 26S proteasome mutants generally show an accelerated cell death phenotype.
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| 5. |
- Boussardon, Clément, et al.
(författare)
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Tissue-specific isolation of Arabidopsis/plant mitochondria - IMTACT (isolation of mitochondria tagged in specific cell types)
- 2020
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Ingår i: The Plant Journal. - : John Wiley & Sons. - 0960-7412 .- 1365-313X. ; 103:1, s. 459-473
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Tidskriftsartikel (refereegranskat)abstract
- 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 inArabidopsis thaliana(Arabidopsis) that involves biotinylation of mitochondria in a tissue-specific manner using transgenic lines expressing a synthetic version of theOM64(Outer Membrane 64) gene combined withBLRPand theBirAbiotin ligase gene. Tissue specificity is achieved with cell-specific promoters (e.g.CAB3andSUC2). 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.
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| 6. |
- Boussardon, Clément, et al.
(författare)
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Tissue-specific isolation of tagged Arabidopsis plastids
- 2023
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Ingår i: Current protocols. - : John Wiley & Sons. - 2691-1299. ; 3:2
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Liebsch, Daniela, et al.
(författare)
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Metabolic control of arginine and ornithine levels paces the progression of leaf senescence
- 2022
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Ingår i: Plant Physiology. - : Oxford University Press. - 0032-0889 .- 1532-2548. ; 189:4, s. 1943-1960
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Przybyla-Toscano, Jonathan, et al.
(författare)
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Gene atlas of iron-containing proteins in Arabidopsis thaliana
- 2021
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Ingår i: The Plant Journal. - : Blackwell Publishing. - 0960-7412 .- 1365-313X. ; 106:1, s. 258-274
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Röhricht, Helene, et al.
(författare)
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Mitochondrial ferredoxin-like is essential for forming complex I-containing supercomplexes in Arabidopsis
- 2023
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Ingår i: Plant Physiology. - : Oxford University Press. - 0032-0889 .- 1532-2548. ; 191:4, s. 2170-2184
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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