A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.
The hemiparasitic plant Phtheirospermum japonicum is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyse the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoterreporter, and further analysed the subcellular localization, the enzymatic function, and contribution to inducing hypertrophy by studying CRISPR-Cas9 induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed isopentenyltransferases activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a was also identified in the related weed Striga hermonthica. With PjIPT1a, we identified the IPT enzyme that induces CK responses in Phtheirospermum japonicum-infected Arabidopsis roots. We propose that PjIPT1a exemplifies how parasitismrelated functions evolve through gene duplications and neofunctionalization.
The hemiparasitic plant Phtheirospermum japonicum is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyse the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analysed the subcellular localization, the enzymatic function, and contribution to inducing hypertrophy by studying CRISPR-Cas9 induced Phtheirospermum mutants. PjIPT1a was specifically expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed isopentenyltransferases activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a with shared characteristics was also identified in the related weed Striga hermonthica. With PjIPT1a we identified a bona fide parasitism gene. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.
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