Heat-Stable COR (Cold-Regulated) Proteins Associated with Freezing Tolerance in Spinach

While most soluble proteins are coagulated by heating at 100°Cfor 10 minutes, some highly hydrophilic COR (Cold-regulated)proteins remain soluble in aqueous solution (Lin et al. 1990).We report here changes in levels of heat-stable proteins andtheir mRNAs during cold acclimation of spinach (Spinacia oleraceaL.). We analyzed heat-stable proteins and the heat-stable translationproducts from poly(A)⁺RNA generated in a wheat germ system.Heat-stable COR proteins with molecular masses of 140 kDa and85 kDa (CORs 140 and 85), were detected in the leaves of cold-acclimatedplants. Increased levels of CORs 140 and 85 correlated withthe development of freezing tolerance during cold acclimation.Interestingly, CORs 140 and 85 accumulated specifically in theleaves and stems and not in the roots of the cold-acclimatedplants. Consistent with this observation, freezing tolerancewas also induced in leaves and stems, but not in roots. Thesedata strongly suggest that CORs 140 and 85 are closely associatedwith freezing tolerance. Accumulation of COR 85 was also inducedby exogenous ABA, drought, and wounding. The possible rolesof CORs 140 and 85 in plants acclimating to low temperatureis given attention. (Received June 11, 1992; Accepted September 1, 1992)     

Polypeptide Composition of Envelopes of Spinach Chloroplasts: Two Major Proteins Occupy 90% of Outer Envelope Membranes

Outer and inner envelope membranes of spinach chloroplasts wereisolated using floatation centrifugation followed by sedimentationsucrose density gradient centrifugation after disruption ofintact chloroplasts by freezing and thawing. Two major fractionswith buoyant densities of 1.11 and 1.08 g cm^–3 and a minorfraction with a density of 1.15 g cm^–3 were obtained.They were identified as innei and outer envelope and thylakoidfractions, respectively, by analyzing their polypeptide compositionby high-resolution SDS-PAGE and the N-terminal sequences oftheir protein components. Due to the refinement of the isolation procedure, most of theribulose-l,5-bisphosphate carboxylase/oxygenasc (RuBisCO), whichhad always been observed as a contaminant, was eliminated fromthe outer envelope fraction. Application of high-resolutionSDS-PAGE revealed that this fraction was rich in the low-molecular-massouter envelope protein, E6.7 [Salomon et at. (1990) Proc. Natl.Acad. Sci. USA 87: 5778] and a protein with a molecular massof 15 kDa which is homologous to the 16 kDa outer envelope proteinof pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94:9504]. The two proteins account for 90% of the total proteinspresent in outer envelope membranes. Proteins which are suggestedto function in translocation of nuclear-encoded polypeptideswere not identified in the envelopes from spinach in the presentstudy. Differences in the protein composition of outer envelopemembranes arc discussed based on the developemental stages ofchloroplasts. ¹Present address: Biological Function Section, Kansai AdvancedResearch Center, Communications Research Laboratory, Ministryof Posts and Telecommunications, Kobe, Hyogo, 651-24 Japan.     

Genetic analysis of the accumulation of COR14 proteins in wild (Hordeum spontaneum) and cultivated (Hordeum vulgare) barley

The cold-regulated (COR14) protein of 14 kDa is a polypeptide accumulated under low-temperature conditions in the chloroplasts of barley leaves. In H. vulgare the COR14 antibody cross-reacts with two proteins, with a slightly different relative molecular weight around the marker of 14.4 kDa, referred to as COR14a and COR14b (high and low relative molecular weight, respectively). In a collection of H. spontaneum genotypes a clear polymorphism was found for the corresponding COR proteins. While some accessions showed the same COR pattern as cultivated barley, in 38 out of 61 accessions examined the COR14 antibody cross-reacted with an additional coldregulated protein with a relative molecular weight of about 24 kDa (COR24). The accumulation of COR24 was often associated with the absence of COR14b; the relationship between the COR14b/COR24 polymorphism and the adaptation of H. spontaneum to different environments is discussed. By studying COR14 accumulation in cultivated barley we have found that the threshold induction-temperature of COR14a is associated with the loci controlling winter hardiness. This association was demonstrated by using either a set of 30 cultivars of different origin, or two sets of frost-tolerant and frost-sensitive F1 doubled-haploid lines derived from the cross Dicktoo (winter type) x Morex (spring type). These results suggest that the threshold induction-temperature of COR14a can be a potential biochemical marker for the identification of superior frostresistant barley genotypes.     

Accumulation of a High Molecular Weight Protein during Cold Hardening of Wheat (Triticum aestivum L.)

Electrophoretic patterns of soluble protein fractions from cold-tolerantwinter wheats (Triticum aestivum L. cv. Frederick and cv. Norstar)and cold-sensitive spring wheat (T. aestiaum L. cv. Glenlea)were analysed in hardened and unhardened plants. One and two-dimensionalgel electrophoresis analysis reveals that cold hardening conditionsinduce changes in the soluble protein patterns. The most importantis the accumulation of a high molecular weight protein in therange of 200 kDa. This protein accumulated at higher concentrationin cold-tolerant cultivars compared to the coldsensitive onesuggesting a correlation between the degree of freezing toleranceand the accumulation of this specific protein. In addition,the intensity of three protein bands (mol wt 48, 47 and 42 kDa)increased while that of five others (mol wt 93, 89, 80, 67 and63 kDa) decreased during hardening. These changes occured inthe three cultivars suggesting that they are part of the metabolicadjustments in response to low temperature rather than a specificchange associated with the development of cold hardiness. (Received April 23, 1987; Accepted June 5, 1987)     

A Prolyl Endoproteinase that Acts Specifically on the Extrinsic 18-kDa Protein of Photosystem II: Purification and Further Characterization

An endoproteinase, which specifically cleaves the Pro12-Leu13bond of the extrinsic 18-kDa protein of PSII, was purified fromPSII membranes of spinach. The presence of 0.05% (w/v) Tween20 and 1 M NaCl was essential for maintenance of proteolyticactivity during the purification. The molecular mass of theenzyme was estimated to be 95 kDa by gel-filtration chromatography.Active fractions contained a polypeptide of 165 kDa that wasconverted into diffusely stained polypeptides of 54 kDa uponreduction with dithiothreitol. The K_m of the 18-kDa proteinin the proteolytic reaction was 0.3 µM. Inhibition ofthe proteolysis by compounds that contain prolyl bonds revealedthat both a prolyl bond and a positive charge are necessaryfor interaction with the proteinase, but some other structuralfactor(s) must also be involved in the high-affinity interactionbetween the proteinase and the 18-kDa protein. Reconstitutionof NaCl-treated PSII membranes with the 23-kDa protein and/orthe 18-kDa protein revealed that the 18-kDa protein was notcleaved by the proteinase when the substrate protein was functionallyassociated with the membranes. A comparison of the propertiesof the proteinase with those of a proline-specific endopeptidasefrom Flavobacterium suggests that these enzymes are quite differentin terms of substrate specificity. (Received December 13, 1993; Accepted March 24, 1994)     

Evidence from Crosslinking for a Close Association of the Extrinsic 33 kDa Protein with the 9.4 kDa Subunit of Cytochrome b 559 and the 4.8 kDa Product of the psb I Gene in Oxygen-Evolving Photosystem II Complexes from Spinach

Oxygen-evolving photosystem II complexes from spinach, whichlack light-harvesting chlorophyll a/b proteins, were treatedwith a bifunctional crosslinking reagent, hexamethylene-diisocyanate.Identification of crosslinked proteins with antisera raisedagainst various constituent proteins of the oxygen-evolvingPS II complex showed that the extrinsic 33 kDa protein is locatedless than 11 Å from the 9.4 kDa subunit of cytochromeb 559 and the 4.8 kDa product of psb I gene. (Received October 14, 1991; Accepted February 6, 1992)     

A mechanistic model of COR15 protein function in plant freezing tolerance: integration of structural and functional characteristics

Plants as sessile organisms are strongly challenged by environmental stresses. Many plants species are able to cold-acclimate, acquiring higher freezing tolerance upon exposure to low but non-freezing temperatures. Among a plethora of adaptational processes, this involves the accumulation of cold regulated (COR) proteins that are assumed to stabilize and protect cellular structures during freezing. However, their molecular functions are largely unknown. We recently reported a comprehensive study of 2 intrinsically disordered cold regulated chloroplast proteins, COR15A and COR15B from Arabidopsis thaliana. They are necessary for full cold acclimation. During freezing, they stabilize leaf cells through folding and binding to chloroplast membranes. Contrary to evidence from in-vitro experiments, they play no role in enzyme stabilization in vivo. Elucidating these major functional and structural characteristics and estimation of protein abundance allow us to propose a detailed model for the mode of action of the two COR15 proteins.     

Purification, Characterization and cDNA Cloning of the 200 kDa Protein Induced by Cold Acclimation in Wheat

We have purified to homogeneity the 200 kDa protein inducedspecifically by low temperature in wheat (Triticum aestivumL.). The boiling solubility of the protein has been used asa main step in the purification procedure. Amino acid compositionindicates that the 200 kDa has a compositional bias for glycine(11.4%), threonine (13.3%), and alanine (22.0%). Using oligonucleotideprobes, we have isolated a clone (pWcs200) from a cold-acclimatedwinter wheat cDNA library. Northern analysis demonstrated thatthe expression of the corresponding gene was specifically upregulatedby low temperature. Southern analysis showed that the gene organizationand the relative copy number were identical in two cultivarsdiffering in their capacity to develop freezing tolerance. Proteinsequence and immunological analyses indicate that this proteinshares similar features with the 50 kDa protein induced duringcold acclimation of wheat. The two proteins are boiling-soluble,and possess similar repeated elements. These elements may beimportant for the development of freezing tolerance. We haveshown that the 200 kDa protein is the largest member of a familyof immunologically-related cold-induced proteins in wheat. Expressionof pWcs200 in E. coli yielded a product of around 200 kDa, indicatingthat the clone contains most of the coding region for this protein. (Received August 18, 1992; Accepted October 14, 1992)     

Changes in patterns of protein synthesis during haustorial development of Striga hermonthica (Del.) Benth. seedlings

This study focuses upon the developmental transition of theparasitic plant Striga hermonthica from its freeliving state(germinated seedling) to its parasitic state after developmentof an infection organ: the haustorium. A new method has beendeveloped that allows the production of gram quantities of germinatedand haustorially-induced Striga seedlings, thereby facilitatingbiochemical and molecular analysis of haustorial induction.Water-soluble proteins have been extracted from germinated seeds(stage A) and seedlings treated with 2,6-dimethoxy-p-benzoquinone(2,6-DMBQ) to induce haustorium (stage B). Samples were analysedby two-dimensional polyacrylamide gel electrophoresis and quantitativeas well as qualitative differences could be observed. In particulara group of four highly abundant acidic proteins (molecular weight39 kDa, pl 5.1, 5.3, 5.3, 5.6) and three other proteins (molecularweight 12 kDa, pl 6.9; 17 kDa, pl 4.4; 17 kDa, pl 4.45) wereseen in stage A while at least four proteins (molecular weight21.5 kDa, pl 6.4; 21.5 kDa, pl 6.3; 31 kDa, pl 5.1; 34 kDa,pl 6.2) were present in greater abundance in stage B. In orderto compare watersoluble protein with newly synthesized proteinpatterns, mRNAs from the two stages of development were isolatedand cell-free translation products analysed by 2-D PAGE. Two-Dgels of cell-free translation products showed the appearanceof six proteins in stage B (molecular weight ranging from 10to 35 kDa) and the presence of three acidic proteins in stageA with one protein (molecular weight 40 kDa) very similar insize to the triplet of proteins in the water-soluble protein2-D gels. Key words: Striga hermonthica (Del.) Benth., haustorium, 2-D PAGE, 2,6-DMBQ, translation in vitro     

Isolation and Characterization of the Cytochrome bf Complex from Whole Thylakoids, Grana, and Stroma Lamellae Vesicles from Spinach Chloroplasts

The cytochrome bf complex was isolated from spinach thylakoids,and also from separated grana and stroma lamellae vesicles,by a procedure involving NaBr washing, detergent treatment andcentrifugation in sucrose gradients. The resulting complex fromall three types of membranes, were almost completely devoidof chlorophyll and carotenoids. The complexes have kinase activitytowards histone III-S and contain a 64 kDa protein claimed tobe a kinase. Electrophoretic analyses indicate that the complexesare in dimeric form and composed of six polypeptides with molecularmasses of 34/33, 23, 20, 17, 12 and 4 kDa. The complexes containtwo moles cytochrome b₆ per mole cytochrome f and one mole RieskeFeS. The 17 kDa and 4 kDa polypeptides are the so called subunit4 and 5 respectively. The 12 kDa protein was identified as plastocyaninby immunoblotting. Plastocyanin and the 4 kDa protein were presentin the cytochrome bf complex even after a second repeated sucrosedensity gradient centrifugation. The sucrose gradient sedimentation pattern was different forthe grana and stroma lamellae complexes. The complex from thestroma lamellae arrives at a higher density than the grana complex.Furthermore, the gradient centrifugation diagram of the stromalamellae consists of one main peak while the diagram of thegrana complex shows two peaks. There is significantly more plastocyaninand 4 kDa protein in the bf complex isolated from stroma lamellaethan from grana. In addition there is a 15 kDa protein in thecomplex isolated from the grana vesicles. Immunoblot analysisafter crosslinking indicated that the 4 kDa protein and theplastocyanin are associated in the cytochrome bf complex. Theoxidoreductase activity is higher (about 50%) in the cytochromebf complex from the grana than from the stroma lamellae fraction.We suggest that a difference in composition of the cytochromebf complex between the two membranes might be important in theregulation of cyclic and non cyclic electron flow. ¹Present address: Department of Plant Physiology II, Universityof Warsaw, 00 927 Warsaw, Poland     

Expression in Escherichia coli of the Extrinsic 18-kDa Protein of Photosystem II of Spinach

A cDNA encoding the precursor for the 18-kDa protein of PSIIof spinach was expressed in Escherichia coli. When the celllysate was incubated at 7°C, the precursor was degradedby proteases of E. coli to a polypeptide of 18 kDa (P18) thatconsisted of the mature protein moiety plus the last four residuesof the transit peptide. P18 was able to reconstitute the water-oxidizingcomplex of NaCl-treated PSII membranes supplemented with the23-kDa protein. Moreover, P18 was cleaved by the prolyl endoproteinaseof spinach specifically at the Pro-12-Leu-13 bond, as was theauthentic 18-kDa protein. These properties of P18 indicate thatthe present expression system is potentially useful for studiesof the substrate specificity of the endoproteinase, as wellas of the structure-function relationships of the 18-kDa protein. (Received November 12, 1994; Accepted January 11, 1995)     

The Isolation and Characterization of a Cytochrome b6f Complex from the Cyanobacterium Spirulina sp.

A cytochrome b₆f complex was isolated and purified from Spirulinasp. The complex was solubilized with n-heptyl ß-D-thioglucosideand chromatographed on a DEAE-Toyopearl 650M column. The purifiedcomplex contained a small amount of chlorophyll and carotenoid.At least four polypeptides were present in the complex: cytochromef (29 kDa), cytochrome b₆(23 kDa), iron-sulfur protein (ISP,23 kDa), and a 17 kDa polypeptide. Each polypeptide was separatedfrom the complex treated with 2-mercaptoethanol or urea. Theabsorption spectra of cytochrome b₆ and cytochrome f were similarto those of Anabaena and spinach as expected. The complex wasactive in supporting ubiquinol-cytochrome c oxidoreductase activity.Fifty percent inhibition of the activity was accomplished by1 µM dibromothymoquinone (DBMIB). The K_m values for ubiquinol-2and cytochrome c (horse heart) were 5.7 µM and 7.4 µM,respectively. (Received August 15, 1988; Accepted November 14, 1988)     

Cold-Induced Alterations in Plasma Membrane Proteins That are Specifically Related to the Development of Freezing Tolerance in Cold-Hardy Winter Wheat

The objective of this study was to identify plasma membraneproteins that are specifically induced by cold acclimation inwheat (Triticum aestivum L.). Two cultivars with a marked differencein the genetic ability to cold-acclimate, namely, spring wheat(cv. Chinese Spring) and winter wheat (cv. Norstar), were usedas the experimental material. After four weeks of growth ina cold chamber, the freezing tolerance in the shoots of winterwheat increased to –18°C, whereas it increased onlyto –8°C in the shoots of spring wheat. In the caseof roots from both cultivars, freezing tolerance increased onlyslightly after the growth in the cold environment. Cold acclimationinduced remarkable changes in the electrophoretic patterns ofplasma membrane proteins which depended on both the cultivarand the tissue examined. Levels of polypeptides with molecularmasses from 22 to 31 kDa decreased in both the root and shootplasma membranes from both cultivars. Among these polypeptides,levels of those of 28 and 26 kDa decreased abruptly after oneweek of cold acclimation. By contrast, levels of polypeptidesof 89, 83, 52, 23, 18 and 17 kDa increased specifically in theshoots of winter wheat. The increases in the levels of the 23-,18- and 17-kDa polypeptides were proportional to the developmentof freezing tolerance. Freeze-fracture electron microscopy ofplasma membranes from shoot cells revealed that the number ofintramembrane particles on the fracture faces decreased markedlyin winter wheat after cold acclimation, but to a lesser extentin spring wheat. These results suggest that the plasma membranesmight undergo molecular reorganization during cold acclimation. ¹Contribution no. 3709 from the Institute of Low TemperatureScience, Hokkaido University.     

Codeinone reductase isoforms with differential stability,efficiency and product selectivity in opium poppy

Codeinone reductase (COR) catalyzes the reversible NADPH‐dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3‐O‐demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O‐demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR‐B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1–1.4 and COR‐B) were used to determine sequence–function relationships that influence product selectivity. Biochemical characterization and site‐directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.     

Discrimination by immunological analysis between two 33–34 kDa polypeptides involved in photosynthetic oxygen evolution

A 34 kDa protein, which is modified in a mutant of Scenedesmus obliquus with impaired oxygen evolution and decreased levels of managese, has been equated to a hydrophilic spinach 33 kDa protein. In the present study, we show immunological evidence that these two proteins are not identical. Scenedesmus wild type, mutant and revertant were analyzed by western blotting using antibodies against the spinach 33 kDa protein. The antibodies did not react with the 34 kDa protein, but with a 30 kDa polypeptide unaltered in the mutant. Thus, in Scenedesmus, and probably also in higher plants, there are two distinct proteins with similar molecular weights, both of which are essential for oxygen evolution and possibly also for managese binding.     

Mobilization of nitrogen reserves during regrowth of defoliated Trifolium repens L. and identification of potential vegetative storage proteins

Although it is well established that carbon reserves contributeto shoot regrowth of leguminous forage species, little informationis available on nitrogen reserves except in Medicaqo sativaL. and Trifolium subterraneum L. In this study, reserves werelabelled with ¹⁵N to demonstrate the mobilization of endogenousnitrogen from roots and stolons to regrowing leaves and newstolons during 24 d of regrowth in white clover (Thfolium repensL.). About 55% and 70%, respectively, of the nitrogen contentsof these organs were mobilized to support the regrowth of leaves.During the first 6 d, nitrogen in regrowing leaves came mainlyfrom N reserves of organs remaining after defoliation. Afterthese first 6 d of regrowth, most of the shoot nitrogen wasderived from exogenous nitrogen taken up while the contributionof nitrogen reserves decreased. After defoliation, the buffer-solubleprotein content of roots and stolons decreased by 32% duringthe first 6 d of regrowth. To identify putative vegetative storageproteins, soluble proteins were separated using SDS-PAGE ortwo-dimensional electrophoresis. One protein of 17.3 kDa instolons and two proteins of 15 kDa in roots seemed to behaveas vegetative storage proteins. These three polypeptides, initiallyfound at high concentrations, decreased in relative abundanceto a large extent during early regrowth and then were accumulatedagain in roots and stolons once normal growth was re-established. Key words: White clover, regrowth, ¹⁵N-labelled, vegetative storage proteins, electrophoresis     

Molecular Sizes of the Catalytic Units of Oxygen Evolution and the Reaction Center in Photosystem II of Spinach Thylakoids

Target analysis of the PS II reaction in spinach thylakoidsshowed that the respective molecular masses of the catalyticunits for oxygen evolution and the reaction center are about120 kDa and 250 kDa based on a kinetic separation of the tworeaction rates. The size of the oxygen-evolving enzyme agreedwith that determined for the PS II preparation from a thermophiliccyanobacterium by the same means [Nugent and Atkinson (1984)FEBS Lett. 170: 89]. Single hit-inactivation of oxygen evolutionand the PS II reaction center units indicates that each functionis driven by a structurally assembled unit. (Received August 6, 1984; Accepted December 17, 1984)