Chlorophyll Biosynthesis: Spectrofluorometric Estimation of Protoporphyrin IX, Mg-Protoporphyrin and Protochlorophyllide

Abstract

The metal ion-induced inhibition of tetrapyr-role biosynthesis was studied in the cyanobacterium Anacystis nidulans. The accumulation of protoporphyrin and Mg proto-porphyrin due to the effect of Co2' and Mn2+ treatment, respectively , pointed to two different sites of inhibition. Chlorophyll biosynthesis is inhibited by iron deficiency induced by a number of metals (1)-cobalt and manganese in particular. The mechanism is considered to be competition for an active site where iron is necessary for functional integrity. The photosynthetic pigment apparatus of blue-green algae contains both chlorophyll and phycobiliproteins. The chromophore of the latter is also a tetrapyrrole, albeit a linear one (2). After nitrate starvation the blue-green algae are capable of regenerating their pigment apparatus, and in the process, several fluorescent tetrapyrroles, intermediates in tetrapyrrole biosynthesis, are detectable (3). In the present paper we show that manganese and cobalt, both inhibitors of chlorophyll biosynthesis, are acting at different stages of tetrapyrrole biosynthesis, and we shall identify the steps in biosynthesis where inhibition occurs. METHODS Anacystis nidulans (strain IU 625) was maintained on agar slants enriched with medium C of Kratz and Myers (4) and illuminated by fluorescent tubes (3,000 lux). When maintained in liquid medium C, the cells were grown in glass tubes at 38°C with an illumination of 10,000 lux and were supplied with 95% air/5% CO2. Nitrate starvation was induced by suspending the algal cells in nitrate-deficient culture medium. For regeneration experiments, the cells were collected and resuspended in whole medium C. Fluorescence measurements were carried out using a Per-kin Elmer MPF 3 spectrofluorimeter. Both excitation and observation slits were set at 6-nm bandwidth. Fluorescence spectra were not corrected for the spectral sensitivity of the apparatus because only relative changes in fluorescence are discussed. Absorption spectra were recorded by a UNICAM SP 1800 UV spectrophotometer, using the opal glass method of Shi-bata et al. (5). Phycocyanin/chlorophyll ratios were calculated from the in vivo absorption spectra (6). Pigment Extraction. A 3-ml algal suspension was mixed with 15 ml of acetone/0.1 M NH4 OH, (9:1 (vol/vol) at 0°C and centrifuged at 39,000 x g for 10 min (7). The supernatant was extracted once with 10 ml and twice with 5 ml of n-hexane. The pooled hexane extracts were back-extracted with 5 ml of fresh 75% aqueous acetone fraction. The pigments in the acetone extract were transferred to ether before chromatography. This was achieved by adding 1/17 of its volume of saturated NaCl and 1/70 of its volume of 0.5 M KH2PO4. The mixture was extracted once with 10 ml of per-oxide-free ether and then twice with 5 ml of ether. The combined ether extracts were concentrated under N2. The water/ acetone residue was extracted three times with 5 ml of ether. This final ether phase was concentrated under N2. All procedures were carried out in the dark. Chromatography. The pigment...

Photoregulation of the Greening Process of Wheat Seedlings Grown in Red Light*

“…Heat killed enzyme was obtained by keeping supernatant in boiling water bath for 10 min. Proto IX was estimated by spectrofluorometry (Hukmani and Tripathy, 1992;Tewari and Tripathy, 1999).…”

“…The fluorescence spectra of Hexane Extracted Acetone Residue solvent mixture (HEAR) preparation (Hukmani and Tripathy, 1992) were recorded in ratio mode in SLM-8000 (SLM Instruments, USA) spectrofluorometer having a photon-counting device. Rhodamine B was used in the reference channel as a quantum counter.…”

“…Emission spectra E400, E420, E440 from HEAR were recorded from 580 to 700 nm and were corrected for photomultiplier tube response. Pchlide contents were calculated (Hukmani and Tripathy, 1992).…”

Assembly of Synthetic Locked Phycocyanobilin Derivatives with Phytochrome in Vitro and in Vivo in Ceratodon purpureus and Arabidopsis

“…The wavelength for exciting light was set to 633 nm, and the emission was recorded through a 670-nm band pass filter. This setup is specific for chlorophyll, and precursors such as protochlorophyllide, which has an emission maximum at 631 nm, would not be detected (Mattheis and Rebeiz, 1975;Hukmani and Tripathy, 1992). Apical cells were selected arbitrarily through the normal optics of the microscope.…”

Differential distribution of chlorophyll biosynthetic intermediates in stroma, envelope and thylakoid membranes in Beta vulgaris

“…Excitation spectra were recorded in ratio mode (channel A/C) having slit width of excitation monochromator set at 4 nm and that of emission monochromator set at 8 nm. Using appropriate equations concentrations of Proto IX, MPE, MV Pchlide and DV Pchlide were quantified (Rebeiz et al 1975;Hukmani and Tripathy 1992). Low-temperature spectra were taken by freezing the sample at 77 K using liquid nitrogen in a Dewar cuvette.…”