Margareta Ryberg

Identification of four universal protochlorophyllide forms in dark-grown leaves by analyses of the 77 K fluorescence emission spectra

Abstract Low-temperature (77 K) fluorescence emission spectra of dark-grown leaves from 12 different plant species were measured, corrected and analysed at different stages of development. The second and fourth derivative of the spectra indicated the peak positions of possible spectral bands, which were used for resolution of the fluorescence spectra into gaussian components. Based on the intensities of the gaussian components, statistical analyses were performed to determine a possible connection between the main peaks and their respective long-wavelength vibrational sublevels. Four universal protochloro-phyll(ide) forms were distinguished, with the main emission bands (half-bandwidths parentheses) at 633 (14.5), 645 (14.3), 657 (10.5) and 670 (20.1) nm respectively. Their vibrational sublevels were found at 693 (20.8), 710 (17.3), 726 (17.7) and 740 (21.0) nm respectively. The spectral characteristics of the different protochlorophyll(ide) forms are discussed in relation to their possible molecular structures and interactions with the surrounding membrane components.

The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bodies in wheat (Triticum aestivum L.).

A fraction of highly purified prolamellar bodies was isolated from etioplasts of wheat (Triticum aestivum L. cv. Starke II, Weibull), as previously described by Ryberg and Sundqvist (1982, Physiol. Plant., 56, 125–132). Studies on the protein composition revealed that only one major polypeptide of an apparent molecular weight of 36000 is present in the fraction of prolamellar bodies. This polypeptide was identified as the NADPH-protochlorophyllide oxidoreductase. The highest specific activity of the enzyme in etiolated leaf tissue was confirmed to be in the fraction of prolamellar bodies.

THE FORMATION OF A SHORT-WAVELENGTH CHLOROPHYLLIDE FORM AT PARTIAL PHOTOTRANSFORMATION OF PROTOCHLOROPHYLLIDE IN ETIOPLAST INNER MEMBRANES

Abstract— Partial phototransformation caused the formation and successive accumulation of spectral forms of chlorophyllide in etioplast inner membrane fractions from wheat (Triticum aestivum L. cv. Walde), Low‐temperature fluorescence emission and circular dichroism spectra showed the formation of two chlorophyllide forms with emission maxima at 683 and 694 nm, respectively. The light dependent accumulation of the two forms differed in prolamellar body (PLB) and prothylakoid (PT) fractions. The 694 nm form was preferentially found in PLB fractions which before irradiation were characterized by a 657 nm emitting protochlorophyllide form and a regular PLB structure. The 683 nm form accumulated to a higher extent in PT fractions which before irradiation contained mainly shortwavelength protochlorophyllide forms. The results indicate that at least two photoactive protochlorophyllide forms must be considered. The major part of protochlorophyllide, with a fluoresence emission maximum at 657 nm, is transformed to the 694 nm emitting chlorophyllide first at high light doses. A minor part, revealed only after Gaussian resolution, has a fluorescence emission maximum at 645 nm and was transformed by low light doses to chlorophyllide emitting at 683 nm.

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins’ largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.

PHOTOTRANSFORMATION OF AGGREGATED FORMS OF PROTOCHLOROPHYLLIDE IN ISOLATED ETIOPLAST INNER MEMBRANES

Abstract— Irradiation of an etioplast inner membrane fraction caused the transformation of two photoactive spectroscopically different protochlorophyllide forms into two chlorophyllide forms. A weak light flash, 6% of a saturating flash, preferentially caused the formation of a short wavelength chlorophyllide form absorbing at 672 nm and emitting at 676 nm. A saturating flash resulted in the formation of the 684 nm absorbing form of chlorophyllide with an emission maximum at 698 nm.

Analysis of the 77 K fluorescence emission and excitation spectra of isolated etioplast inner membranes

Abstract Low-temperature fluorescence emission and excitation analyses revealed four discrete spectral forms of protochlorophyllide and/or protochlorophyll in the etioplast inner membranes isolated from leaves of wheat (Triticum aestivum cv. Kosack). The emission spectra were recorded with various excitation wavelengths between 400 and 500 nm. The excitation spectra were recorded for fluorescence emitted at various wavelengths between 620 and 740 nm. The spectra were analysed by a combination of various computer-aided methods including differential analysis, gaussian resolution and topological plots. The four pigment forms can be characterized by their different excitation (Ex) and emission (Em) bands. Adding the satellite bands of the Qy(0-0) transitions, the emission vibrational (Emv) bands, with correlated amplitudes gave the following delineation: Ex436-Em633-Emv690, Ex443-Em645-Emv711, Ex451-Em657-Emv727, and Ex463-Em671-Emv740. The differences in the spectral properties of the four pigment forms are discussed in relation to possible differences in their structural arrangement, association to NADPH-protochlorophyllide oxidoreductase and localization in prolamellar bodies and/or prothylakoids.

The Shibata shift; effects of in vitro conditions on the spectral blue-shift of chlorophyllide in irradiated isolated prolamellar bodies

Abstract The spectral blue-shift of chlorophyllide (Chlide) in flash irradiated isolated prolamellar bodies (PLBs) of dark-grown wheat ( Triticum aestivum ) was analysed. The fluorescence emission at −196°C of Chlide varied depending on the composition of the medium. NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) activity was taken as an indication that a general protein denaturation had not occurred. The results indicate that the spectral blue-shift of Chlide is in part owing to conformational changes of the Chlide-Pchlide reductase complexes induced by the phototransformation of Pchlide to Chlide. The Chlide spectral blue-shift decreased with increasing concentrations of sucrose or glycerol. At high concentration of glycerol (87%) there was neither a Chlide blue-shift nor any esterification of Chlide, while the phototransformation of Pchlide to Chlide was not affected. Phototransformation of Pchlide to Chlide may induce changes in the pigment-Pchlide reductase interactions that cannot be transduced into a molecular rearrangement of the enzyme protein in the presence of high concentrations of osmotic agents. EDTA and Ca 2+ caused effects which in part could be explained as an enhanced disaggregation of the oligomeric Chlide-Pchlide reductase complexes. Addition of BSA enhanced the blue-shift of Chlide even at high sucrose concentrations. It also inhibited a reformation of Pchlide-Pchlide reductase complexes and reduced the esterification of Chlide. The effect of BSA on the blue-shift was suppressed by addition of NADPH. The possibility that BSA interacted with the Chlide-Pchlide reductase complexes is discussed.

ADP/ATP and protein phosphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes.

The effects of modulated ADP/ATP and NADPH/NADP+ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, protochlorophyllide, and NADPH-protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat (Triticum aestivum) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of protochlorophyllide and the reformation of phototransformable protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable protochlorophyllide to POR-associated phototransformable protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP+ ratio is suggested.

Plastid microtubule-like structures in wheat are insensitive to microtubule inhibitors

The effects of microtubule inhibitors on the spectral properties of leaves of wheat (Triticum aestivum L. cv. Walde) and on the presence of plastid microtubule-like structures (MTLS) during etioplast to chloroplast transformation were examined. Amiprophos-methyl (APM, 0.1 mM), fed to leaf sections of 7-day-old dark-grown wheat, reduced the ration of phototransformable to non-phototransformable proto-chlorophyllide (PChlide), decreased the rate of the Shibata shift, and inhibited chlorophyll accumulation and grana stacking. The spectral properties of isolated etioplasts were not affected by APM. Colchicine (10 mM), fed to leaf sections, inhibited greening but had no effect on the PChlide ratio or the Shibata shift. MTLS were still visible on electron micrographs after treatment with APM or colchicine at frequencies similar to controls. A third inhibitor, vinblastine, had no effect on the spectral properties of non-irradiated or irradiated etiolated leaves except at concentrations that produced visible tissue damage before the irradiation. The effects of APM and colchicine may reflect inhibitions of respiration and protein synthesis, respectively. It is concluded that MTLS are insensitive to microtubule inhibitors and thus are probably not composed of tubulin.

Analysis of protochlorophyllide reaccumulation in the phytochrome chromophore-deficient aurea and yg-2 mutants of tomato by in vivo fluorescence spectroscopy

The 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.