Jürgen M. Schmitt

Proteins from frost-hardy leaves protect thylakoids against mechanical freeze-thaw damage in vitro.

We have isolated protein fractions from cold-acclimated, frost-hardy cabbage (Brassica oleracea L.) and spinach (Spinacia oleracea L.) leaves which protect isolated thylakoids from non-hardy spinach against mechanical membrane rupture during an in-vitro freeze-thaw cycle. No protective activity was found in similar preparations from non-hardy leaves. The proteins protected the membranes from damage by reducing their solute permeability during freezing and by increasing their expandability during thawing. The proteins act by increasing the resistance of the membranes against the osmotic stress to which they are exposed during a freeze-thaw cycle. In the absence of cryoprotectants this stress results in membrane rupture.

Chloroplast and Cyanobacterial Genomes, Genes and RNAs: a Compilation

The organization and expression of plastid genomes are among the most extensively studied rid& in plant molecular biology. Restriction endonuclease cleavage site maps have been constructed for the plastid genomes of many plant species. Genes for the rRNAs, for most of the tRNAs and for several proteins have been located on these physical maps. Furthermore, the nucleotide sequences of many of these genes, and/or the corresponding RNAs, have been determined. This article, which is an up-date of the information found in Bohnert et al., 1982 and Crouse et al., 1984, compiles the information on plastid genomes, genes and RNAs which has appeared in the literature. Some highlights of the data, along with the literature citations, are presented in tableform. In addition, related information on cyanobacteria is included. Table 1 lists the plastid genomes for which physical maps have been constructed, and indicates the genes which have been mapped. Sequenced genes for rRNAs, tRNAs and proteins are compiled in Tables 2, 3 and 4, respectively. Gene

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1–3-linked fucose.

Antibodies against individual thylakoid membrane proteins as molecular probes to study chemical and mechanical freezing damage in vitro

Abstract The release of proteins and the loss of biochemical activities under mechanical and chemical stresses during freezing of isolated thylakoid membranes were investigated, using polyacrylamide gel electrophoresis, single radial immunodiffusion and the measurement of cyclic photophosphorylation. Antibodies against purified proteins derived from the stromal (coupling factor CF1, ferredoxin-NADP+ reductase) and the lumenal side (plastocyanin) of the membrane vesicles were used as probes. Low initial solute concentrations were employed to generate mechanical stress. Chemical stresses were manipulated by varying the molar ratios of cryotoxic to cryoprotective solutes at high initial solute concentrations. Constant low amounts of ferredoxin-NADP+ reductase were lost from the membranes during freezing, irrespective of the composition of the suspending media. Damage at high initial osmolalities was accompanied by the release of CF1, which was influenced by the ratio of potentially cryotoxic to cryoprotective solutes, as demanded by the colligative theory of membrane cryopreservation. CF1 release and loss of cyclic photophosphorylation were linearly correlated at different ratios of salt to sucrose. However, the correlation data revealed that CF1 release could account for only part of the observed cryoinjury. Plastocyanin release was predominant at low initial osmolalities and was not influenced by the chemical composition of the suspending media. This indicates mechanical damage by membrane rupture. Under these circumstances loss of plastocyanin and loss of cyclic photophosphorylation were linearly correlated. Loss of photophosphorylation could be prevented by the addition of up to 1.2 mg plastocyanin/ml prior to freezing. It could also be ameliorated to a large extent by raising the phenazine methosulfate concentration in the test assay from 30 to 230 μM. This indicates that the membranes are able to reseal after rupture, maintaining a proton gradient upon illumination and that it is the loss of plastocyanin from their lumen that inhibits cyclic photophosphorylation.

DIRECT SCREENING OF A SMALL GENOME : ESTIMATION OF THE MAGNITUDE OF PLANT GENE EXPRESSION CHANGES DURING ADAPTATION TO HIGH SALT

SummaryMesembryanthemum crystallinum (common ice plant), a facultative halophyte with a genome size of 393 000 kb, was used to estimate the magnitude of changes in gene expression in response to environmental stress by excess salt. Such treatment induces a water-conserving pathway of carbon assimilation (CAM) which is, at least in part, transcriptionally controlled. From a genomic library, 200 phage containing approximately 3200 kb (0.8% of the genome) were randomly selected. The inserts in these clones could be divided into four classes ranging from highly repetitive DNA (class I clones) to single-copy DNA (class IV clones). The inserts of the 166 clones of classes II to IV were digested with various restriction enzymes and the fragments were analyzed by hybridization with radioactively labelled mRNA isolated from stressed and unstressed leaves. We found that a total of ∼ 140 DNA fragments hybridized with the RNA probe. Among those, several differentially regulated transcripts were observed. Stress-dependent fluctuation of mRNA abundance was verified by Northern analyses: one mRNA, not detectable in unstressed leaves, appeared in stressed leaves, while steady-state levels of three transcripts decreased during stress. All regulated signals are derived from low abundance mRNAs, which may be missed during screening of cDNA libraries. We conclude from these results that, for the entire genome, on the order of more than one hundred genes are differentially regulated in response to salt stress.

Age-dependent induction of pyruvate, orthophosphate dikinase in Mesembryanthemum crystallinum L.

A full-length transcript for pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1), has been characterized from Mesembryanthemum crystallinum. Under salt stress or with increasing age, this plant shows a transition from C3 to Crassulacean acid metabolism (CAM). The PPDK plays a central role in gluconeogenesis during the light phase of CAM. The transcript is 3165 bases in length with a single open reading frame of 2739 nucleotides specifying a protein of molecular mass 103098, including a transit peptide of mass 7902 for chloroplast import. The protein shares 44–77% sequence identity with PPDK from C4-plants and microorganisms. Known functional and regulatory amino acids are conserved. Southern-type hybridizations indicated one copy or very few closely related copies of the gene per haploid genome. We investigated the induction of PPDK at the mRNA and protein levels, using the well characterized induction of a CAM-form of phosphoenol pyruvate carboxylase (PEPCase) as internal standard. During wilting of excised leaves PEP-Case mRNA amounts increased strongly within 8 h. Under these conditions amounts of PPDK mRNA remained constant. Re-hydrating leaves from previously stressed plants led to a decrease in PEPCase and PPDK mRNA amounts. During salt stress, no correlation between PEPCase and PPDK was observed. Analysis of plants of different ages indicated that, even in well-watered plants, PPDK-specific protein and mRNA increased when the plants reached a certain age. In old plants, salt stress failed to further increase PPDK mRNA or protein levels. We conclude that PPDK and PEPCase, which are both required for CAM to be functional, are controlled by different regulatory mechanisms in the intact plant.

Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts

Intact chloroplasts and vacuoles were isolated from mesophyll protoplasts of barley. The chloroplasts occupied about 15% of the cellular volume and contained 75% of the protein, whereas the vacuoles occupied about 80% of the volume and contained less than 4% of total cellular protein. Contamination of the vacuolar fraction by foreign protein is included in these values. Chlorophyll was absent from the vacuolar fraction, but less than 1% of several extra-vacuolar marker proteins were still present. The vacuoles contained hydrolytic enzymes. Several of them (α-mannosidase, α-galactosidase, N-acetylglucosaminidase) were soluble, whereas part of the activity of others semimented with the tonoplasts during centrifugation. Attached proteins could be released from the membranes during freezing in the presence of NaCl. One-dimensional gel electrophoretic separation of soluble vacuolar proteins under non-denaturing conditions yielded more than 10 protein bands. A comparative analysis was performed of thylakoids and vacuoles which were subfractionated into tonoplasts and soluble vacuolar constituents. Sodium dodecyl sulfate gel electrophoresis separated about 15 polypeptides of the soluble fraction which reacted with silver reagent. The tonoplast fraction yielded about 20 bands. A similar number of bands was observed when vacuoles incubated with the 14C-labelled SH-reagent N-ethylmaleimide were analysed for radioactive polypeptides. Silverstaining of the polypeptides and their SH-content did not correlate. Several polypeptides of the vacuolar fraction had molecular weights very similar to the molecular weights of known chloroplast proteins. However, with the exception of the two subunits of ribulose-1,5-bisphosphate carboxylase, contamination of the vacuolar fraction by chloroplast proteins could be ruled out as a possible cause of the close correspondence. The lipophilic carboxylic-group reagent N,N′-dicyclohexylcarbodiimide ([14C]DCCD) reacted with several polypeptides of thylakoids and tonoplasts. However, the labelling patterns were different. The most heavily labelled polypeptide of thylakoids was the 8-kDa polypeptide of the basal part of the coupling factor CF0. Tonoplast polypeptides heavily labelled with [14C]DCCD had molecular weights of 24, 28, and 56 kDa. The vacuolar 8-kDa polypeptide remained unlabelled.

Cryotoxicity of antifreeze proteins and glycoproteins to spinach thylakoid membranes — comparison with cryotoxic sugar acids

We have used thylakoids from spinach (Spinacia oleracea L.) chloroplasts to test the effects of antifreeze proteins (AFP) from the starry flounder (Platichthys stellatus; AFP-SF) and from the antarctic eel pout (Austrolycichthys brachycephalus; AFP-AB), and antifreeze glycoproteins (AFGP) from the antarctic fish Dissostichus mawsoni on biological membranes during freezing. Freeze-thaw damage, measured as the release of the lumenal protein plastocyanin from the thylakoid vesicles, was strongly increased in the presence of all proteins tested. Measurements of the time dependence of plastocyanin release in a simplified artificial chloroplast stroma medium showed that all the fish proteins increased damage during the initial rapid phase while only AFGP increased plastocyanin release during the linearly time dependent slow phase. A slow plastocyanin release is also seen in the absence of freezing. It is increased by the presence of AFGP and AFP-AB, but not by AFP-SF. In order to distinguish between the contribution of the polypeptide and the carbohydrate part of AFGP on freeze-thaw damage we investigated the effects of galactose and N-acetylgalactosamine. While galactose was protective, N-acetylgalactosamine increased the rate of plastocyanin release in an artificial stroma medium at -20 degrees C. It had no effect on the rapid phase of damage and was also ineffective at 0 degree C. The same was found for several other sugar derivatives (N-acetylglucosamine, gluconic acid, glucuronic acid, galacturonic acid). From these data we conclude that the increased plastocyanin release during the rapid phase of freeze-thaw damage is a function of the polypeptide part of AFGP. The increased rate of plastocyanin loss at longer incubation times both at 0 degree C and at -20 degrees C may be mediated by the N-acetylgalactosamine moiety of the AFGP, but is strongly amplified by the polypeptide.

Colligative and non-colligative freezing damage to thylakoid membranes

Abstract When spinach thylakoid membranes were frozen in vitro in solutions containing constant molar ratios of cryotoxic to cryoprotective solute, maintenance of functional integrity strongly depended on initial osmolarities. Optimum cryopreservation of cyclic photophosphorylation was observed when the membranes were suspended in solutions of intermediate osmolarities (approx. 50–100 mM NaCl, 75–150 mM sucrose). Both higher and lower initial osmolarities were found to result in decreased cryopreservation. In the absence of added salt, more than 100 mM sucrose were needed for full cryopreservation of the membranes. When thylakoids were frozen in solutions containing low concentrations of NaCl (2 mM), the ratio of sucrose to salt necessary to give full protection was high (up to 50). When the salt concentration was about 60 mM, ratios as low as 1.5 were sufficient for maintaining membrane integrity. This ratio increased again, as the initial NaCl concentration was increased beyond 60 mM. During freezing, proteins dissociated from the membranes, and the amount of the released proteins was correlated linearly with inactivation of photophosphorylation. The gel electrophoretic pattern of proteins released at low initial osmolarities differed from that of proteins released at high initial osmolarities. Cryopreservation was also found to depend on membrane concentration. Concentrated membrane suspensions suffered less inactivation than dilute suspensions. The protective effect of high membrane concentrations was particularly pronounced at high initial solute concentrations. It is proposed that damage at low initial osmolarities is caused predominantly by mechanical stress and by osmotic contraction/expansion. Damage at high initial osmolarities is thought to be caused mainly by solute effects. Under these conditions, both the final volume of the unfrozen solution in coexistence with ice and the membrane concentration affect membrane survival by influencing the extent of the loss of membrane components through dissociation reactions. Membrane protection by sugars is caused by colligative action under these circumstances.

The solute permeability of thylakoid membranes is reduced by low concentrations of trehalose as a co-solute.

The different efficiencies of sucrose and trehalose in protecting isolated spinach (Spinacia oleracea L.) thylakoids against freeze-thaw damage is quantitatively related to their ability to reduce the solute loading of the vesicles during freezing. In the present paper we show that this effect is based on a reduction of the solute permeability of the membranes. Permeability was measured with 14C-labeled glucose at temperatures between 0 and 10 degrees C. Glucose permeability was reduced by both sucrose and trehalose, with trehalose effective at much lower concentrations than sucrose. An analysis of the temperature dependence of glucose permeability in the presence and absence of trehalose revealed that a 50% reduction in permeability resulted from a 10% increase in activation energy and a 30% decrease in activation entropy. Using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH), we found that the reduced permeability of the membranes in the presence of trehalose was unaccompanied by a reduction in lipid fluidity. This also excluded the possibility of a solute-induced liquid crystalline to gel phase transition. A reduced partitioning of the hydrophobicity-sensitive dye merocyanine 540 into thylakoids and into membranes containing 50% digalactosyldiacylglycerol in the presence of trehalose as compared to sucrose and glucose showed that the lipid headgroup region of these membranes became less accessible for solutes. No significant difference in merocyanine partitioning in the presence of trehalose as compared to sucrose or glucose was apparent when monogalactosyldiacylglycerol dispersions or phosphatidylcholine vesicles were investigated.