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Phytochrome chromophore-deficient mutants
No full textPhytochrome chromophore-deficient mutants have been used as phytochrome-deficient plants to study many aspects of plant development. However, there are still a number of important questions to be resolved concerning both the targets and the phenotypic consequences of these mutations. Recently, progress has been made in our understanding of the molecular basis of the chromophore deficiency in these mutants. Biochemical assays for the committed steps of chromophore synthesis have been developed and used to demonstrate that the pcd1 and yellow-green-2 mutants of pea and tomato, respectively, are unable to synthesize biliverdin IX? from heme while pcd2 and aurea are deficient in phytochromobilin synthase activity. This review focuses on how this information can be used to help understand the basis of other chromophore-deficient mutants, such as the hy1 and hy2 mutants of Arabidopsis, and discusses how the phenotype of chromophore-deficient mutants is related to lesions in the chromophore biosynthesis pathway.<br/
Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy
No full textThe aurea and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum) are unable to synthesize the phytochrome chromophore from heme resulting in a block of this branch of the tetrapyrrole pathway. We have previously shown that these mutants also exhibit an inhibition of protochlorophyllide (Pchlide) synthesis and it has been hypothesised that this is due to feedback inhibition by heme on the synthesis of 5-aminolevulinic acid (ALA). In this study we have investigated Pchlide reaccumulation in cotyledons from etiolated wild-type (WT), aurea and yg-2 seedlings using low-temperature fluorescence spectroscopy. WT cotyledons showed two characteristic Pchlide emission maxima at 630 nm (F630) and 655 nm (F655) respectively, while the aurea and yg-2 mutants contained only phototransformable Pchlide F655. Following a white-light flash to WT cotyledons, reaccumulation of phototransformable Pchlide F655 in the first 30 min was absolutely dependent on the presence of Pchlide F630 before the flash. Reaccumulation of Pchlide F630 was not apparent until at least 2 h after the phototransformation. In contrast, Pchlide F630 never accumulated in aurea cotyledons. The relative rates of both Pchlide F655 and total Pchlide synthesis were approximately twice as high in WT compared to aurea. Measurement of ALA synthesis capacity during this period showed that the reduced rate of Pchlide reaccumulation in aurea was due to an inhibition at this step of the pathway. In addition, feeding of ALA resulted in a substantial and equal increase of non-phototransformable Pchlide in both WT and aurea indicating that aurea cotyledons are capable of accumulating high levels of Pchlide that is not associated to the active site of NADPH:Pchlide oxidoreductase (POR). The implications of these results for the mechanism of inhibition of Pchlide synthesis in phytochrome chromophore-deficient mutants and the role of non-phototransformable Pchlide F630 during plastid development are discussed
Holophytochrome assembly. Coupled assay for phytochromobilin synthase in organello
No full textUtilizing an in vitro coupled assay system, we show that isolated plastids from cucumber cotyledons convert the linear tetrapyrrole biliverdin IX alpha to the free phytochrome chromophore, phytochromobilin, which assembles with oat apophytochrome to yield photoactive holoprotein. The spectral properties of this synthetic phytochrome are indistinguishable from those of the natural photoreceptor. The plastid-dependent biliverdin conversion activity is strongly stimulated by both NADPH and ATP. Substitution of the nonnatural XIII alpha isomer of biliverdin for the IX alpha isomer affords a synthetic holophytochrome adduct with blue-shifted difference spectra. These results, together with experiments using boiled plastids, indicate that phytochromobilin synthesis from biliverdin is enzyme-mediated. Experiments where NADPH (and ATP) levels in intact developing chloroplasts are manipulated by feeding the metabolites 3-phosphoglycerate, dihydroxyacetone phosphate, and glucose 6-phosphate or by illumination with white light, support the hypothesis that the enzyme that accomplishes this conversion, phytochromobilin synthase, is plastid-localized. It is therefore likely that all of the enzymes of the phytochrome chromophore biosynthetic pathway reside in the plastid
Making light of it: the role of plant haem oxygenases in phytochrome chromophore synthesis
No full textThe haem oxygenase (HO) enzyme catalyses the oxidation of haem to biliverdin IXa, CO and Fe2+, and performs a wide variety of roles in Nature, including degradation of haem from haemoglobin, iron acquisition and phycobilin biosynthesis. In plants, HOs are required for the synthesis of the chromophore of the phytochrome family of photoreceptors. There are four HO genes in the Arabidopsis genome. Analysis of a mutant deficient in HO1 (the hy1 mutant) has demonstrated that this plastid-localized protein is the major HO in the phytochrome chromophore synthesis pathway. HO2 may also have a minor role in this pathway, but our understanding of the divergent roles of this small gene family is still far from complete
Inactivation of phytochrome- and phycobiliprotein-chromophore precursors by rat liver biliverdin reductase
No full textThe phytochrome chromophore precursor, 3E-phytochromobilin, and the phycobiliprotein chromophore precursors, 3E-phycocyanobilin and 3E-phycoerythrobilin, are enzymatically converted to novel rubinoid products by purified rat liver biliverdin reductase. Phytochromobilin and phycocyanobilin are particularly good substrates for biliverdin reductase with Km and Vmax values very similar to those of the natural substrate, biliverdin IX alpha. Phycoerythrobilin is the least preferred of the three bilin substrates. 1H NMR spectroscopy of phycocyanorubin, the product of phycocyanobilin catalysis by biliverdin reductase, and comparison of absorption spectra of all three rubinoid products reveal that the C10 methine bridge is selectively reduced by biliverdin reductase without altering the A-ring ethylidene substituent. In vitro phytochrome assembly experiments demonstrate that the phytorubin products do not form photoactive adducts with recombinant apophytochrome. These results suggest that ectopic expression of biliverdin reductase in plants will prevent assembly of the functional photoreceptor and thus will potentially alter light-mediated plant growth and development
Purification of plasma membrane from wheat leaves and characterization of the associated vanadate-sensitive Mg2+ -ATPase activity
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(3Z)- and (3E)-phytochromobilin are intermediates in the biosynthesis of the phytochrome chromophore
No full textUsing a high performance liquid chromatography (HPLC)-based assay, we have demonstrated that isolated oat etioplasts convert the linear tetrapyrrole biliverdin IX alpha to (3E)-phytochromobilin, the proposed precursor of the chromophore of the plant photoreceptor phytochrome. In addition to (3E)-phytochromobilin, the synthesis of a second phytochromobilin was detected by its ability to functionally assemble with recombinant oat apophytochrome A. The structure of this new pigment has been determined to be the 3Z isomer of phytochromobilin by absorption and 1H NMR spectroscopy. Like (3E)-phytochromobilin, assembly of HPLC-purified (3Z)-phytochromobilin with apophytochrome yielded a holoprotein that is spectrally indistinguishable from native oat phytochrome A. However, the postchromatographic conversion of (3Z)- to (3E)-phytochromobilin appears to be responsible for this result. Kinetic HPLC analyses have demonstrated that (3Z)-phytochromobilin is synthesized prior to the 3E isomer by oat etioplasts. We therefore propose that (3Z)-phytochromobilin is the immediate product of biliverdin IX alpha reduction by the enzyme phytochromobilin synthase. This implicates the presence of an isomerase that catalyzes the conversion of (3Z)- to (3E)-phytochromobilin, the immediate precursor of the phytochrome A chromophore
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