Caleosins were originally described as one of the two major protein components of storage lipid bodies in the seeds of higher plants, the other being oleosins. In contrast with oleosins, caleosins have a single calcium-binding EF-hand domain plus several potential phosphorylation sites and have been hypothesised as playing a role in lipid-body formation and possibly mobilisation in seeds. In Arabidopsis, there are six functional caleosin genes, two of which encode seed-specific proteins while the other isoforms are expressed in a variety of vegetative and reproductive tissues. More recently, seed caleosins have been shown to have a peroxygenase activity but the function of this was uncertain. This study describes the characterisation of a specific membrane-bound caleosin isoform, termed Clo-3 in Arabidopsis and Brassica spp, that appears to be present in all plant tissues and is responsive to a variety of biotic and abiotic stresses. Bioinformatic analysis reveals that similar caleosin-like genes/proteins are present in all vascular plants, as well as in nonvascular plants such as mosses, and even in single-celled algae. Intriguingly, caleosin-like genes are also present in the genomes of most fungi described to date, with the surprising exception of the yeasts. In order to understand the function of caleosins in plants, a detailed structural and functional analysis of this novel class of protein is reported here. Biochemical studies demonstrate that the Clo-3 isoform binds calcium (one atom per molecule), can be phosphorylated most likely, by a casein kinase 2 (CK2) protein kinase, and has putative peroxygenase activity. In addition to biochemical data, microscopy analysis shows that Clo-3 may be located both on the endoplasmic reticulum and chloroplastid envelope membranes. specifically the chloroplast envelope. Biochemical evidence of cell membrane localisation is also presented. Protease digestion experiments show that the membrane bound Clo-3 has a Type I transmembrane orientation, where its N-terminal domain faces the lumen of microsomes while the C-terminal is on the cytosolic face. Such an orientation is common for receptors or proteins that may be activated by signalling molecules. The Clo-3 gene and its encoded protein are each upregulated by salt and drought stresses and by abscisic acid (ABA) treatment. Reverse genetics using RNAi knockdown mutants demonstrate specific transcription factors involved in regulating Clo-3 during different stresses. Peroxygenase activities of Clo-3 enriched microsomes were higher following salt stress. Although the data is representative of potentially many peroxygenases, it does provide indirect evidence that Clo-3 abundance increases and/or catalytic activity is induced during stress. The study also presents evidence of the response of Clo-3 to biotic stress and related signalling molecules. Arabidopsis Clo-3 is highly responsive to the phytohormone salicylic acid, to the salicylic acid synthetic analogue DCINA, the biotic signalling molecule hydrogen peroxide, and to infection by the common fungal pathogen of Brassicas, Leptosphaeria maculans (Phoma), while experiments utilising the non-expressor of pathogenesis related protein 1 (npr1) knockout mutant plant demonstrates Clo-3 response to salicylic acid (SA) is chiefly via npr1 translocation to the nucleus. The type of peroxygenase epitomised by Clo-3 is similar to those involved in the formation of epoxy alcohols from fatty acid hydroperoxides. The latter are a class of oxylipins that are seen in fungal infection, and also play a role in various aspects of fungal spore development including sporulation and a role in cuticle synthesis. As such, Clo-3 in Arabidopsis and possibly similar caleosins in other species might play roles in oxylipin signalling pathways that are involved in a protective role during both biotic and abiotic stress responses.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:503374 |
| Date | January 2009 |
| Creators | Partridge, Mark |
| Publisher | University of South Wales |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://pure.southwales.ac.uk/en/studentthesis/investigation-of-the-roles-of-a-membranebound-caleosin-in-higher-plants(fd83462e-5c65-4faf-b785-fc05cac37c33).html |
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