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- Betti, Marco, et al.
(författare)
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Manipulating photorespiration to increase plant productivity : recent advances and perspectives for crop improvement
- 2016
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 67:10, s. 2977-2988
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Forskningsöversikt (refereegranskat)abstract
- Recycling of the 2-phosphoglycolate generated by the oxygenase reaction of Rubisco requires a complex and energy-consuming set of reactions collectively known as the photorespiratory cycle. Several approaches aimed at reducing the rates of photorespiratory energy or carbon loss have been proposed, based either on screening for natural variation or by means of genetic engineering. Recent work indicates that plant yield can be substantially improved by the alteration of photorespiratory fluxes or by engineering artificial bypasses to photorespiration. However, there is also evidence indicating that, under certain environmental and/or nutritional conditions, reduced photorespiratory capacity may be detrimental to plant performance. Here we summarize recent advances obtained in photorespiratory engineering and discuss prospects for these advances to be transferred to major crops to help address the globally increasing demand for food and biomass production.
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- 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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- 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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- 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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| 7. |
- 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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| 8. |
- 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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| 10. |
- Brouwer, Bastiaan, et al.
(författare)
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In response to partial plant shading, the lack of phytochrome A does not directly induce leaf senescence but alters the fine-tuning of chlorophyll biosynthesis
- 2014
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 65:14, s. 4037-4049
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Tidskriftsartikel (refereegranskat)abstract
- Phytochrome is thought to control the induction of leaf senescence directly, however, the signalling and molecular mechanisms remain unclear. In the present study, an ecophysiological approach was used to establish a functional connection between phytochrome signalling and the physiological processes underlying the induction of leaf senescence in response to shade. With shade it is important to distinguish between complete and partial shading, during which either the whole or only a part of the plant is shaded, respectively. It is first shown here that, while PHYB is required to maintain chlorophyll content in a completely shaded plant, only PHYA is involved in maintaining the leaf chlorophyll content in response to partial plant shading. Second, it is shown that leaf yellowing associated with strong partial shading in phyA-mutant plants actually correlates to a decreased biosynthesis of chlorophyll rather than to an increase of its degradation. Third, it is shown that the physiological impact of this decreased biosynthesis of chlorophyll in strongly shaded phyA-mutant leaves is accompanied by a decreased capacity to adjust the Light Compensation Point. However, the increased leaf yellowing in phyA-mutant plants is not accompanied by an increase of senescence-specific molecular markers, which argues against a direct role of PHYA in inducing leaf senescence in response to partial shade. In conclusion, it is proposed that PHYA, but not PHYB, is essential for fine-tuning the chlorophyll biosynthetic pathway in response to partial shading. In turn, this mechanism allows the shaded leaf to adjust its photosynthetic machinery to very low irradiances, thus maintaining a positive carbon balance and repressing the induction of leaf senescence, which can occur under prolonged periods of shade.
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