Moss survival through in situ cryptobiosis after six centuries of glacier burial.

Cryptobiosis is a reversible ametabolic state of life characterized by the ceasing of all metabolic processes, allowing survival of periods of intense adverse conditions. Here we show that 1) entire moss individuals, dated by 14C, survived through cryptobiosis during six centuries of cold-based glacier burial in Antarctica, 2) after re-exposure due to glacier retreat, instead of dying (due to high rates of respiration supporting repair processes), at least some of these mosses were able to return to a metabolically active state and remain alive. Moss survival was assessed through growth experiments and, for the first time, through vitality measurements. Future investigations on the genetic pathways involved in cryptobiosis and the subsequent recovery mechanisms will provide key information on their applicability to other systematic groups, with implications for fields as divergent as medicine, biodiversity conservation, agriculture and space exploration.

The amino acid sequence of a major protein component in the light harvesting complex of the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum.

A 7.5-kDa protein has been isolated from chlorosomes of Chlorobium limicola f. thiosulfatophilum and the complete primary structure determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The 74-residue protein shows great homology to a similar protein of unknown function which has been isolated from Pelodictyon luteolum but otherwise no significant homology to other proteins can be found. The possible role of the protein in the structure and function of the chlorosome is discussed.

Circular dichroism of green bacterial chlorosomes.

Positive and negative bands in previously measured circular dichroism (CD) spectra of Chlorobium limicola chlorosomes appeared to be sign-reversed relative to those of Chloroflexus aurantiacus chlorosomes in the 740-750 nm spectral region where bacteriochlorophyll (BChl) c absorbs maximally. It was not clear, however, whether this difference was intrinsic to the chlorosomes or was due to differences in the procedures used to prepare them. We therefore repeated the CD measurements using chlorosomes isolated from both Cb. limicola f. thiosulfatophilum and Cf. aurantiacus using the method of Gerola and Olson (1986, Biochim. Biophys. Acta 848: 69-76). Contrary to the earlier results, both types of chlorosomes had very similar CD spectra, suggesting that both have similar arrangements of BChl c molecules. The previously reported difference between the CD spectra of Chlorobium and Chloroflexus chlorosomes is due to the instability of Chlorobium chlorosomes, which can undergo a hypsochromic shift in their near infrared absorption maximum accompanied by an apparent inversion in their near infrared CD spectrum during isolation. Treating isolated chlorosomes with the strong ionic detergent sodium dodecylsulfate, which removes BChl a, does not alter the arrangement of BChl c molecules in either Chloroflexus or Chlorobium chlorosomes, as indicated by the lack of an effect on their CD spectra.

A new bacteriochlorophyll a-protein complex associated with chlorosomes of green sulfur bacteria.

Chlorosomes were prepared from Chlorobium limicola f. thiosulfatophilum by sucrose density gradient centrifugation. Cells broken in the presence of 2 M NaSCN yielded three chlorosome fractions in the gradient: low density (no sucrose), medium density (approx. 18% sucrose), and high density (approx. 26% sucrose). All fractions were stable at any chlorosome concentration. Cells broken in the absence of 2 M NaSCN also yielded three fractions, but only the high-density fraction contained stable chlorosomes. The medium-density chlorosomes were stable only when highly concentrated. Upon dilution, bacteriochlorophyll (BChl) c was degraded to bacteriopheophytin c and concomitantly a band at 794 nm (BChl a) was revealed. Two 794-nm fractions were observed with the same densities as low- and medium-density chlorosomes. The protein composition of the 794-nm fractions was similar to that of the stable chlorosome fractions. All showed a 4-5 kDa (Mr) protein as a major component, but no trace of the 40-kDa protein characteristic of the water-soluble BChl a-protein of green sulfur bacteria. BChl a was present in all types of chlorosomes, in stable chlorosomes the BChl c/BChl a ratio was approx. 90. A special BChl a-protein (794 nm) inside the chlorosome is postulated to mediate energy transfer from BChl c to the water-soluble BChl a-protein in the baseplate.

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids.

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent Km for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.

Effects of trypsin and cations on chloroplast membranes.

A mild tryptic digestion of chloroplast membranes eliminates the effects of saturating concentrations of cations (3 to 5 millimolar MgCl(2)) on chlorophyll fluorescence yield, membrane stacking, and photosystem II photochemical efficiency in spinach. At the same time, the negative surface potential of the membranes is increased (by trypsin) as revealed by studies with 9-aminoacridine. High concentrations of cations (25 to 100 millimolar MgCl(2)) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations added after trypsin treatment do not increase the photosystem II efficiency. It is concluded that the "diffuse electrical layer" hypothesis of Barber et al. (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily explains the effect of trypsin in eliminating the influence of saturating concentrations of cations on chlorophyll fluorescence yield and membrane stacking. However, the effect on photosystem II photochemical efficiency seems to require another mechanism.

Evidence for a structural role for chlorophyll in chlorophyll-protein complexes.

1. Chymotrypsin treatment of spinach chloroplast membranes does not change the electrophoretic mobility of either chlorophyll-protein complex 1 or 2. 2. The extraction of lipids with 80% acetone after treatment of the membranes with chymotrypsin reveals that the polypeptide components of both chlorophyll-protein complexes had been extensively digested. The extraction of carotenes with petroleum ether under the same conditions does not change the electrophoretic mobility of the chlorophyll-protein complexes. 3. Fluorescence polarisation studies of chlorophyll-protein complex 2 reveal that the chymotrypsin digestion of this complex does not result in changes of mutual orientation or distance apart of chlorophyll a, chlorophyll b or carotenoid. 4. Two polypeptide components have been detected after lipid extraction of electrophoretically purified chlorophyll-protein complexes 1 and 2. The SDS molecular weights are 24 000 and 27 000 for complex 2, and 68 000 and 64 000 for complex 1. 5. We conclude that chlorophyll performs an important structural function in both chlorophyll-protein complexes.

Partition zone penetration by chymotrypsin, and the localization of the chloroplast flavoprotein and photosystem II.

1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.

Studies on Cation-induced Thylakoid Membrane Stacking, Fluorescence Yield, and Photochemical Efficiency.

Trypsin digestion of photosynthetic membranes isolated from spinach (Spinacia oleracea L.) leaves eliminates the cation stimulation of chlorophyll fluorescence. High concentrations of cations protect the fluorescence yield against trypsin digestion, and the cation specificity for this protection closely resembles that required for the stimulation of fluorescence by cations. Trypsin digestion reverses cation-induced thylakoid stacking, and the time course of this effect seems to parallel that of the reversal of cation fluorescence. High concentrations of cations protect thylakoid stacking and cation-stimulated fluorescence alike. The cation stimulation of photosytem II photochemistry remains intact after trypsinization has reversed both cation-induced thylakoid stacking and fluorescence yield. It is concluded that cation-stimulated fluorescence yield, and not the cation stimulation of photosystem II photochemistry, is associated with thylakoid membrane stacking.

Inhibition of photosynthesis by azide and cyanide and the role of oxygen in photosynthesis.

Cyanide and azide inhibit photosynthesis and catalase activity of isolated, intact spinach (Spinacia oleracea) chloroplasts. When chloroplasts are illuminated in the presence of CN(-) or N(3) (-), accumulation of H(2)O(2) is observed, parallel to inhibition of photosynthesis. Photosynthetic O(2) evolution is inhibited to the same extent, under saturating light, whether CO(2) or phosphoglycerate is present as electron acceptor.The illumination of chloroplasts with CN(-) or N(3) (-) inactivates the NADPH- and ATP-dependent phosphoglycerate reduction. This enzyme system can be reactivated by dithiothreitol. In reconstituted, envelope-less chloroplasts, the phosphoglycerate-dependent and the ribose 5-phosphate-dependent O(2) evolution are inhibited to the same extent, while electron transport to NADP is unaffected.It is concluded that the inhibition of photosynthesis by CN(-) and N(3) (-) is due to H(2)O(2) accumulation, which is a consequence of catalase inhibition.The inhibition of phosphoglycerate reduction, but not of CO(2) reduction, is abolished under conditions where ATP is available in excess of NADPH (low light, supply of ATP). This is taken as an indication that electron flow from photosystem I is diverted to O(2) (Mehler reaction, which produces H(2)O(2)) when the unavailability of ATP is limiting the rate of reoxidation of NADPH. The Mehler reaction is considered a physiological process supplying ATP for photosynthesis.