Spectrofluorometric estimation of intermediatesof chlorophyll biosynthesis: Protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide

Hukmani, Pinki; Tripathy, Baishnab C.

doi:10.1016/s0003-2697(05)80021-1

Cited by 30 publications

(11 citation statements)

References 10 publications

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“…However, in Arabidopsis, Mg-ProtoIX was the only intermediate to significantly accumulate following treatment with norflurazon to initiate plastid-to-nucleus communication ( Figure 2D) (Strand et al, 2003). The emission peaks detected from the dissolved tetrapyrrole standards by the confocal microscope were as expected between 620 and 640 nm for ProtoIX and Mg-ProtoIX and between 580 and 600 nm for its methyl ester (Hukmani and Tripathy, 1992). Thus, confocal laser scanning spectroscopy could be used to visualize and discriminate between Mg chelated forms of tetrapyrroles.…”

Section: Visualization Of Tetrapyrroles Using Confocal Laser Scanningsupporting

confidence: 52%

In Vivo Visualization of Mg-ProtoporphyrinIX, a Coordinator of Photosynthetic Gene Expression in the Nucleus and the Chloroplast

et al. 2007

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The photosynthetic apparatus is composed of proteins encoded by genes from both the nucleus and the chloroplast. To ensure that the photosynthetic complexes are assembled stoichiometrically and to enable their rapid reorganization in response to a changing environment, the plastids emit signals that regulate nuclear gene expression to match the status of the plastids. One of the plastid signals, the chlorophyll intermediate Mg-ProtoporphyrinIX (Mg-ProtoIX) accumulates under stress conditions and acts as a negative regulator of photosynthetic gene expression. By taking advantage of the photoreactive property of tetrapyrroles, Mg-ProtoIX could be visualized in the cells using confocal laser scanning spectroscopy. Our results demonstrate that Mg-ProtoIX accumulated both in the chloroplast and in the cytosol during stress conditions. Thus, the signaling metabolite is exported from the chloroplast, transmitting the plastid signal to the cytosol. Our results from the Mg-ProtoIX over-and underaccumulating mutants copper response defect and genome uncoupled5, respectively, demonstrate that the expression of both nuclear-and plastid-encoded photosynthesis genes is regulated by the accumulation of Mg-ProtoIX. Thus, stress-induced accumulation of the signaling metabolite Mg-ProtoIX coordinates nuclear and plastidic photosynthetic gene expression.

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Section: Visualization Of Tetrapyrroles Using Confocal Laser Scanningsupporting

confidence: 52%

In Vivo Visualization of Mg-ProtoporphyrinIX, a Coordinator of Photosynthetic Gene Expression in the Nucleus and the Chloroplast

et al. 2007

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“…Similarly, when thylakoids were added to the supernatant fraction of the plastid lysate, Proto IX synthesis from ALA was completely inhibited. Under the experimental conditions used, inhibition of Proto IX synthesis was not due to quenching of Proto IX¯uorescence by chlorophyllide or chlorophyll (Hukmani and Tripathy 1992). This inhibition was further probed by adding thylakoids (200 lg protein) after termination of the reaction with acetone.…”

Section: Inhibition Of Proto IX Synthesis By Thylakoidsmentioning

confidence: 97%

“…Incubation was carried out for 1 h at 28°C (Tripathy and Rebeiz 1986). The reaction was stopped by adding 1.5 ml of ice-cold 90% acetone, centrifuged at 15,000 rpm (15,900g) for 5 min, and the acetone extract was taken for separation and subsequent¯uorometric estimation of Proto IX (Rebeiz et al 1975;Hukmani and Tripathy 1992).…”

Section: Synthesis Of Proto IX From Alamentioning

confidence: 99%

“…Acetone was extracted with hexane to prepare a hexane-extracted acetone-residue solvent mixture as described before (Hukmani and Tripathy 1992). The hexane-extracted acetone-residue solvent mixture contained Proto IX and other non-esteri®ed porphyrins.…”

Section: Separation Of Pigmentsmentioning

confidence: 99%

“…Excitation and emission slit widths were 4 nm. Using appropriate equations, the concentration of Proto IX was quanti®ed and values expressed as nmol (mg protein) )1 h )1 (Rebeiz et al 1975;Hukmani and Tripathy 1992).…”

Section: Spectro¯uorometrymentioning

confidence: 99%

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Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Manohara

Tripathy

2000

Planta

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Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction.

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Catalytic Function of a Novel Protein Protochlorophyllide Oxidoreductase C of Arabidopsis thaliana

Pattanayak

Tripathy

2002

Biochemical and Biophysical Research Communications

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Spectrofluorometric estimation of intermediatesof chlorophyll biosynthesis: Protoporphyrin IX, Mg-protoporphyrin, and protochlorophyllide

Cited by 30 publications

References 10 publications

In Vivo Visualization of Mg-ProtoporphyrinIX, a Coordinator of Photosynthetic Gene Expression in the Nucleus and the Chloroplast

In Vivo Visualization of Mg-ProtoporphyrinIX, a Coordinator of Photosynthetic Gene Expression in the Nucleus and the Chloroplast

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Catalytic Function of a Novel Protein Protochlorophyllide Oxidoreductase C of Arabidopsis thaliana

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