A novel proteomic approach reveals a role for Mg-protoporphyrin IX in response to oxidative stress.

The presence of genes encoding organellar proteins in different cellular compartments necessitates a tight coordination of expression by the different genomes of the eukaryotic cell. This coordination of gene expression is achieved by organelle-to-nucleus communication. Stress-induced perturbations of the tetrapyrrole pathway trigger large changes in nuclear gene expression. In order to investigate whether the tetrapyrrole Mg-ProtoIX itself is an important part of plastid-to-nucleus communication, we used an affinity column containing Mg-ProtoIX covalently linked to an Affi-Gel matrix. The proteins that bound to Mg-ProtoIX were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with nano liquid chromatography-mass spectrometry (MS)/MS. Thus, we present a novel proteomic approach to address the mechanisms involved in cellular signaling and we identified interactions between Mg-ProtoIX and a large number of proteins associated with oxidative stress responses. Our approach revealed an interaction between Mg-ProtoIX and the heat shock protein 90-type protein, HSP81-2 suggesting that a regulatory complex including HSP90 proteins and tetrapyrroles controlling gene expression is evolutionarily conserved between yeast and plants. In addition, our list of putative Mg-ProtoIX-binding proteins demonstrated that binding of tetrapyrroles does not depend on a specific amino acid motif but possibly on a specific fold of the protein.

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

Stroma, envelope and thylakoid membranes were prepared from chloroplasts isolated from leaves of Beta vulgaris. Out of total plastidic protochlorophyllide, envelope membranes contained 1.5%, thylakoids had the maximum 98.48% and stroma had a trace fraction of 0.02%. Distribution of the Mg-protoporphyrin IX and its monoester was 89.0% in thylakoids, 10.0% in stroma and 1.0% in envelope. A substantial fraction (33.77%) of plastidic protoporphyrin IX was partitioned into stroma. Envelope contained 0.66% and thylakoids had 65.57% of the total plastidic protoporphyrin IX pool. The proportion of monovinyl and divinyl forms of protochlorophyllide was almost similar in intact plastid, thylakoids, and outer and inner envelope membranes suggesting a tight regulation of vinyl reductase enzyme. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma is discussed.

Impairment of the photosynthetic apparatus by oxidative stress induced by photosensitization reaction of protoporphyrin IX.

Treatment with the herbicide acifluorfen-sodium (AF-Na), an inhibitor of protoporphyrinogen oxidase, caused an accumulation of protoporphyrin IX (Proto IX) , light-induced necrotic spots on the cucumber cotyledon within 12-24 h, and photobleaching after 48-72 h of light exposure. Proto IX-sensitized and singlet oxygen ((1)O(2))-mediated oxidative stress caused by AF-Na treatment impaired photosystem I (PSI), photosystem II (PSII) and whole chain electron transport reactions. As compared to controls, the F(v)/F(m) (variable to maximal chlorophyll a fluorescence) ratio of treated samples was reduced. The PSII electron donor NH(2)OH failed to restore the F(v)/F(m) ratio suggesting that the reduction of F(v)/F(m) reflects the loss of reaction center functions. This explanation is further supported by the practically near-similar loss of PSI and PSII activities. As revealed from the light saturation curve (rate of oxygen evolution as a function of light intensity), the reduction of PSII activity was both due to the reduction in the quantum yield at limiting light intensities and impairment of light-saturated electron transport. In treated cotyledons both the Q (due to recombination of Q(A)(-) with S(2)) and B (due to recombination of Q(B)(-) with S(2)/S(3)) band of thermoluminescence decreased by 50% suggesting a loss of active PSII reaction centers. In both the control and treated samples, the thermoluminescence yield of B band exhibited a periodicity of 4 suggesting normal functioning of the S states in centers that were still active. The low temperature (77 K) fluorescence emission spectra revealed that the F(695) band (that originates in CP-47) increased probably due to reduced energy transfer from the CP47 to the reaction center. These demonstrated an overall damage to the PSI and PSII reaction centers by (1)O(2) produced in response to photosensitization reaction of protoporphyrin IX in AF-Na-treated cucumber seedlings.

Biodiversity of hydrogenases in Frankia.

Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.

A hydrogen-evolving enzyme is present in Frankia sp. R43.

The ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes Frankia sp. R43 and Frankia sp. KB5. To further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. Under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) Frankia sp. R43 but not Frankia sp. KB5 evolved hydrogen,which was not linked to NAD-reducing activity. Immunological analysis of total protein from Frankia sp. R43 and Frankia sp. KB5 using an antiserum raised against Ralstonia eutropha HoxF, recognized an antigen in Frankia sp. R43 but not in Frankia sp. KB5. Immunogold labeling using antibodies raised against the R. eutropha HoxH recognized sites in both hyphae and vesicles of Frankia sp. R43, but not in Frankia sp. KB5. Based on these physiological and immunological findings, we conclude that Frankia sp. R43 has a hydrogen-evolving hydrogenase.

Developmental changes in sub-plastidic distribution of chlorophyll biosynthetic intermediates in cucumber (Cucumis sativus L.).

Four-day-old etiolated cucumber seedlings (Cucumis sativus L.) were transferred to cool-white-fluorescent light (15 mumol m-2 s-1) for 1 h and 24 hours and etiochloroplasts and chloroplasts were isolated from developing cotyledons. Plastids were fractionated to stroma, envelope and thylakoid or inner membranes and the pigment contents of all these different fractions were analysed. In intact cucumber chloroplast protochlorophylide was present in significant amounts whereas protoporphyrin IX and Mg-protoporphyrin plus its monoester were present only in very small quantities. Out of the total chloroplastic protochlorophylide pool 1.0% was partitioned to envelope membranes and 99.0% was partitioned to thylakoids. Stroma had only trace amounts of protochlorophylide. In contrast to chloroplasts, etiochloroplasts had, besides protochlorophylide, significant amounts of other chlorophyll biosynthetic intermediates. In etiochloroplasts, protoporphyrin IX primarily partitioned to inner membranes (59.1%) followed by stroma (37.7%) and envelope (3.21%). The content of Mg-protoporphyrin IX plus its monoester in different subplastidic fractions was 74.4% for inner membranes, 22.58% for stroma and 3.02% for envelope. Protochlorophyllide primarily partitioned to inner membranes (95.79%), followed by envelope (4.15%) and, to a negligible extent (0.06%), into stroma. The sub-plastidic distribution of chlorophyll biosynthetic intermediates in etiochloroplasts was, therefore, different than that of chloroplasts. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma in developing and mature plastids is discussed in relation to chloroplast biogenesis.

Detection of protoporphyrin IX in envelope membranes of pea chloroplasts.

Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)(-1). Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis.