Cold Hardiness and Acclimation Intensity in Flower Buds for Fall Bloom and Spring Bloom Clones in Rhododendron kiusianum, a Dwarf Evergreen Azalea

The relationship between the degree of cold hardiness (supercoolingability of florets) and the acclimation intensity in flowerbuds was investigated in the fall bloom and the spring bloom(typical) clones of Rhododendron kiusianum, a hardy dwarf evergreenazalea. Supercooling ability or exotherm temperature distribution(ETD) in florets was determined by differential thermal analysis(DTA) and the intensity of bud acclimation or the rate of deacclimationwas judged by the changes in ETD profiles resulting from thedehardening temperature treatment. Although the two clone typesshowed no significant differences in ETDs and water contentsin florets, they differed in their rates of bud deacclimation.The flower buds of fall bloom clones generally tend to deacclimatemore quickly than the spring bloom ones throughout the seasons.It is concluded that the degree of cold hardiness in flowerbuds of R. kiusianum does not differ between the fall bloomand spring bloom clones but the intensity of bud acclimationdoes; acclimation intensity is higher in the spring bloom clonesand the rate of deacclimation is greater in the fall bloom ones. (Received October 14, 1985; Accepted February 5, 1986)     

Seasonal Changes in Plasma Membranes and Mitochondria Isolated From Jerusalem Artichoke Tubers. Possible Relationship to Cold Hardiness

Plasma membranes and mitochondria were isolated from Jerusalemartichoke tubers during cold acclimation from September to December.The protein and lipid contents of the membranes were analyzedwith reference to physiological properties of the tubers, especiallycold hardiness. As cold hardiness increased from autumn to winter,the content of phospholipids and sterols on a mg protein basisincreased by 20–30% in plasma membranes, but little changewas observed in mitochondria. Minor changes were observed inthe fatty acid composition of phospholipids either in plasmamembranes or mitochondria. Membrane fluidity, assessed by fluorescentpolarization of 1,6-diphenyl-1,3,5-hexatriene, was found tobe relatively constant in both membranes during the season.One dimensional SDS-polyacrylamide gel electrophoresis revealedseasonal changes in proteins and glycoproteins in plasma membranes,but not in mitochondrial membranes. Plasma membrane ATPase increasedin specific activity from September to December, which was morenoticeable at higher assay temperatures. However, irrespectiveof the season, the plasma membrane ATPase had an inflectionon the slope of the Arrhenius plot around 15C. These resultssuggest that plasma membranes, in contrast to mitochondria,undergo several molecular changes from autumn to winter, whichmay be related to cold acclimation of the tubers. ¹ Contribution No. 2668 from the Institute of Low TemperatureScience. ² Present address: Crop Development Centre, University of Saskatchewan,Saskatoon, Canada S7N 0W0.     

Differences in cold hardiness,carbohydrates, dehydrins and related gene expressions under an experimental deacclimation and reacclimation in Prunus persica

To boost our understanding of a recent outbreak of freezing injury, we sought to confirm distinctive features between the shoot tissues of the peach (Prunus persica) cultivars Daewol and Kiraranokiwami by mimicking unseasonable changes of temperatures that occur in the early spring through repeated deacclimation and reacclimation treatments. Patterns of cold hardiness declined dramatically during the deacclimation and rose during the reacclimation in both cultivars. Our results indicated that ‘Daewol’ possessed higher capacity in response to repeated deacclimation and reacclimation treatments than ‘Kiraranokiwami’. ‘Daewol’ showed more sensitive changes in the carbohydrates in response to warm and low temperatures compared with ‘Kiraranokiwami’. ‘Daewol’ indicated almost similar repeated down‐ and up‐patterns in soluble sugar content in response to repeated deacclimation and reacclimation, whereas it indicated repeated up‐ and down‐patterns in starch content. However, ‘Kiraranokiwami’ showed a progressive increase in the soluble sugar content and a progressive decrease in starch content. Notably, patterns of accumulation of a 60‐kDa dehydrin protein encoded by the PpDhn1 gene were confirmed through western blotting and paralleled fluctuations of cold hardiness in both cultivars. Expression of this dehydrin was weak in both cultivars during deacclimation but its band intensity increased during reacclimation. Changes in related genes (β‐amylase, PpDhn1, PpDhn2 and PpDhn3) were positively correlated with changes in cold hardiness throughout the experiment. Our results indicate that recent repeated warm periods may cause premature deacclimation in the early spring, and that more cold‐tolerant cultivar may be more resilient to freezing injury caused by unstable temperature conditions.     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H⁺ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H⁺-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90–100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase     

Isolation and Characterization of the Plasma Membrane by Two-Phase Partitioning from the Alkalophilic Cyanobacterium Spirulina platensis

Partition in aqueous dextran-polyethylene glycol two-phase systemwas used to isolate the plasma membranes from the alkalophiliccyanobacterium Spirulina platensis. The upper phase containeda colorless membranes obtained in relatively short time, 3–4h. This fraction had a different protein profile than that ofthe thylakoid fraction obtained in the lower phase. It did notcontain cytochrome c-oxidase activity, but retained characteristicMg²⁺-ATPase activity that is sensitive to vanadate, stimulatedby K⁺, and has a pH optimum near 8.5. These data support ourassumption that the upper phase of the gradient consist of theplasma membrane of S. platensis. (Received November 25, 1993; Accepted April 12, 1994)     

Changes in ribosomal patterns and a related membrane fraction during induction of cold hardiness in mimosa epicotyl tissues

Sucrose gradient profiles of polyribosomes from epicotyl tissuesof mimosa seedlings (Albizzia julibrissin Durazzini) were followedthroughout induction of cold hardiness. An unusual fractionappeared in the polyribosome region of sucrose gradients withadvancing stages of cold hardiness. Treatment of this unusualfraction and polyribosome fractions with RNase, Triton X-100and deoxycholate suggests that the unusual fraction consistedof chloroplast or mitochondrial membrane fragments. Free polyribosome patterns were retained throughout inductionof cold hardiness. However, the presence of polyribosomes duringadvancing stages of cold hardiness does not necessarily insureactive protein synthesis. ¹ This investigation was supported in part by National ScienceFoundation Grant GB 8692. ² Contribution from the Missouri Agricultural Experiment Station.Journal Series Number 6253. (Received November 30, 1971; )     

Variations in Cold Resistance among Apple Cultivars during Deacclimation

Coleman, W. K. 1985. Variations in cold resistance among applecultivars during deacclimation.——J. exp. Bot. 36:1159–1171. One-year-old vegetative twig samples from mature, bearing treesof nine apple cultivars were monitored over two years for theirdormancy intensity and relative cold hardiness levels duringthe winter/spring deacclimation period. The apple cultivarsexhibited a consistent response during the dehardening processwhich included a higher initiation temperature for the low temperatureexotherm (LT₂) and the development of an intermediate freezingexotherm (LT₁.). Imperial Red Mac/Antonovka was the hardiestcultivar during the two-year period while Imperial Red Mac/M.111was the most tender. Cortland/Beautiful Arcade and Rogers RedMac/M.111 varied considerably in their relative hardiness responsesfrom year to year. Mid-winter hardiness levels were significantlyand positively correlated with dormancy intensity in the ninecultivars. However, this relationship did not exist when thehardiness indices for late winter or early spring were comparedwith dormancy intensity. An intensive correlation and path analysisof the response of four cultivars (Jersey Mac/M.111, Vista Bella/M.111,Spur Mac/M.111 and Rogers Red Mac/M.111) to previous maximum/minimumair temperatures indicated that past maximum temperature primarilyaffected LT₂ while past minimum temperature affected LT₁. Whenlinear regression equations were fitted to the data, the meanair temperature of 0°C coincided with LT₁ values of —18 °C and LT₂ values of –36°C to –38°Cfor all four cultivars. Correlation analyses between % moisturecontent and LT₁/LT₂ for the four cultivars were often positivebut generally non-significant. Injury in living cells slightlypreceded the initiation temperature of LT₁ and supports theidea that membrane destabilization may be an important and immediateprecursor to intracellular freezing. Key words: Apple, cold hardiness, deacclimation     

Estimation of the Freezing Injury in Flower Buds of Evergreen Azaleas by Water Proton Nuclear Magnetic Resonance Relaxation Times

Temperature dependence of longitudinal relaxation times (T₁)of water protons in flower buds of six azalea species differingin cold hardiness and ecological distribution was investigatedby pulse nuclear magnetic resonance spectroscopy. Thermal hysteresiswas observed for T₁ following a slow freeze-thaw cycle. TheT₁ ratio (the ratio obtained from the difference between theoriginal T₁ value in an unfrozen sample and the final T₁ aftera freeze-thaw treatment, both at 20C, divided by the originalT₁) was closely correlated with the viability of florets innon-acclimated buds of R. kiusianum. If the buds were frozento a lethal temperature and then thawed to 20C, the T₁ ratioincreased. The T₁ ratios of acclimated winter buds for the sixspecies used were correlated with the level of cold hardiness(supercooling ability of florets determined by differentialthermal analysis). The T₁ ratio of deacclimated spring buds,especially those from hardier species, markedly increased uponcooling to a lethal temperature. Species differences observedin acclimated winter buds disappeared upon deacclimation. TheT₁ ratio appears to be related to the viability of florets andthe degree of freezing damage (membrane disruption) in florets. (Received December 28, 1984; Accepted May 24, 1985)     

Quantitative and qualitative changes in carbohydrates associated with spring deacclimation in contrasting Hydrangea species

Cold deacclimation and associated changes in soluble carbohydrates and water status of two Hydrangea species differing in susceptibility to frost injuries was followed under natural conditions. In fully cold hardy plants of H. macrophylla stem freezing tolerance fluctuated in parallel with changes in air temperature, while in a seasonal perspective increased temperatures caused a sigmoid deacclimation pattern in both H. macrophylla and H. paniculata. Timing of deacclimation was approximately synchronized in the two species, but H. paniculata, the hardier species based on mid-winter hardiness, deacclimated faster than H. macrophylla, indicating that deacclimation kinetics were not correlated with mid-winter hardiness. In both species concentrations of soluble sugars decreased during deacclimation and were highly correlated with stem cold hardiness and air temperatures. This suggests that sugar hydrolysis may be an important temperature-driven mechanism of deacclimation in Hydrangea. Accumulation patterns of specific carbohydrates differed between the two species, suggesting that they utilize different strategies to overcome cold. In H. paniculata, deacclimation was associated with an increase in stem water content, which occurred shortly before bud burst and hence may be a prerequisite for leafing out.     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)     

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.     

Alterations in Detergent-Resistant Plasma Membrane Microdomains in Arabidopsis thaliana During Cold Acclimation

Microdomains in the plasma membrane (PM) have been proposedto be involved in many important cellular events in plant cells.To understand the role of PM microdomains in plant cold acclimation,we isolated the microdomains as detergent-resistant plasma membranefractions (DRMs) from Arabidopsis seedlings and compared lipidand protein compositions before and after cold acclimation.The DRM was enriched in sterols and glucocerebrosides, and theproportion of free sterols in the DRM increased after cold acclimation.The protein-to-lipid ratio in the DRM was greater than thatin the total PM fraction. The protein amount recovered in DRMsdecreased gradually during cold acclimation. Cold acclimationfurther resulted in quantitative changes in DRM protein profiles.Subsequent mass spectrometry and Western blot analyses revealedthat P-type H⁺-ATPases, aquaporins and endocytosis-related proteinsincreased and, conversely, tubulins, actins and V-type H⁺-ATPasesubunits decreased in DRMs during cold acclimation. Functionalcategorization of cold-responsive proteins in DRMs suggeststhat plant PM microdomains function as platforms of membranetransport, membrane trafficking and cytoskeleton interaction.These comprehensive changes in microdomains may be associatedwith cold acclimation of Arabidopsis.     

Ultrastructural Changes in Cortical Cells of Apple (Malus pumila Mill.) Associated with Cold Hardiness

Seasonal ultrastructural changes in cortical cells of apple(Malus pumila Mill.) twigs were studied with special referenceto seasonal variations in cold hardiness. The ultrastructuralcharacteristics could be divided into two major sets: one setthat developed during cold acclimation from September to Januaryand one that developed during deacclimation from February toMay. During cold acclimation, the most striking changes weremicrovacuolation and augmentation of the volume of the cytoplasm.At this stage, the cells became temporarily rich in the organellesthat are involved in protein synthesis, such as vesicular endoplasmicreticulum, polysomes, dictyosomes and vesicles. Plastids thatcontained starch granules, protein-lipid bodies and mitochondriawere also abundant. Each nucleus contained relatively loweramounts of heterochromatin and was located in the central portionof the cell. From mid-November until March, plastids aggregatedaround the nucleus, and the formation of "plastid initials"from the mature plastids, as a results of constriction and subsequentpinching off, was frequently observed. In January, when maximumcold-hardiness was achieved, the starch granules in plastidsdisappeared. The second set of ultrastructural changes, whichincluded fusion of vacuoles, was initiated in late February.During deacclimation, the differentiation of vacuoles proceededin the cells, starch granules reappeared in the plastids andorganelles involved in protein synthesis became abundant. Furthermore,vesicular endoplasmic reticulum observed during the autumn andwinter was replaced by smooth endoplasmic reticulum. In mid-May,when cold hardiness decreased to a low level, most of the cellspace was occupied by a large vacuole. The results suggest that seasonal cytological changes are involvedin the changes in the physical and functional properties ofcold-adapted cells. The seasonal replication of vacuoles andcytoplasm may be related to resistance or susceptibility tothe stress-producing effects of the dehydration that occursduring freezing, and the replication of organelles may be associatedwith the metabolism required for cold acclimation or deacclimation. (Received December 3, 1992; Accepted December 28, 1993)     

Vacuolar Membrane Lesions Induced by a Freeze-Thaw Cycle in Protoplasts Isolated from Deacclimated Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The processes of freezing injury in Jerusalem artichoke (Helianthustuberosus L.) tubers were studied using protoplasts isolatedfrom cold-acclimated and deacclimated tubers. Prior to freezing,protoplasts were preloaded with 10 µM fluorescein diacetate(FDA) in an isotonic sorbitol solution. After freeze-thawingat various temperatures, cell viability was evaluated undera fluorescence microscope. In cold-acclimated tubers, more than80% of protoplasts survived freezing to – 20°C. Bycontrast, in deacclimated tubers, the cell survival abruptlydeclined after freezing to temperatures below – 5°C.Thus, freezing tolerance differed significantly between protoplastsisolated from cold-acclimated and deacclimated tubers. Two distincttypes of cell injury, which were caused by either damage toplasma membrane (cell-lysis type) or by damage to the vacuolarmembrane (abnormal-staining type), were observed, dependingon the cold hardiness and freezing temperature. In the cellsof the abnormal-staining type, shrinkage of the central vacuolarspace and simultaneous acidification of the cytoplasmic spacewere characteristically observed immediately before completecell-rehydra-tion during thawing. The decrease in freezing toleranceof protoplasts after deacclimation was suggested to be due mainlyto destabilization of the vacuolar membrane by freeze-induceddehydration stress. ¹Contribution no. 3945 from The Institute of Low TemperatureScience, Hokkaido University. This research was supported inpart by the grant from Japan Society for the Promotion of Science(JSPS-RFTF 96L00602) ²Present address: Tohoku National Agricultural Experiment Station, Morioka, Iwate, 020-01 Japan     

Dynamic thermal time model of cold hardiness for dormant grapevine buds

Background and Aims

Grapevine (Vitis spp.) cold hardiness varies dynamically throughout the dormant season, primarily in response to changes in temperature. The development and possible uses of a discrete-dynamic model of bud cold hardiness for three Vitis genotypes are described.

Methods

Iterative methods were used to optimize and evaluate model parameters by minimizing the root mean square error between observed and predicted bud hardiness, using up to 22 years of low-temperature exotherm data. Three grape cultivars were studied: Cabernet Sauvignon, Chardonnay (both V. vinifera) and Concord (V. labruscana). The model uses time steps of 1 d along with the measured daily mean air temperature to calculate the change in bud hardiness, which is then added to the hardiness from the previous day. Cultivar-dependent thermal time thresholds determine whether buds acclimate (gain hardiness) or deacclimate (lose hardiness).

Key Results

The parameterized model predicted bud hardiness for Cabernet Sauvignon and Chardonnay with an r² = 0·89 and for Concord with an r² = 0·82. Thermal time thresholds and (de-)acclimation rates changed between the early and late dormant season and were cultivar dependent but independent of each other. The timing of these changes was also unique for each cultivar. Concord achieved the greatest mid-winter hardiness but had the highest deacclimation rate, which resulted in rapid loss of hardiness in spring. Cabernet Sauvignon was least hardy, yet maintained its hardiness latest as a result of late transition to eco-dormancy, a high threshold temperature required to induce deacclimation and a low deacclimation rate.

Conclusions

A robust model of grapevine bud cold hardiness was developed that will aid in the anticipation of and response to potential injury from fluctuations in winter temperature and from extreme cold events. The model parameters that produce the best fit also permit insight into dynamic differences in hardiness among genotypes.     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Protein and Lipid Compositions of Isolated Plasma Membranes from Orchard Grass (Dactylis glomerata L.) and Changes during Cold Acclimation

The chemical composition of plasma membrane fractions isolated from orchard grass seedlings (Dactylis glomerata L.) was analyzed and compared with endomembranes. The plasma membrane is characterized by an enrichment of sterols and a lower degree of unsaturation of phospholipids. Steryl glycosides were found to be one of the lipid components of the plasma membrane, but steryl esters and galactolipids were barely detectable. Diphosphatidyl glycerol was characteristically detected in the mitochondrial membrane, but not in the plasma membrane fraction. Plasma mambrane fraction was also characterized by its `lower fluidity' in comparison with the endomembranes. This may be due to the large amount of sterols and the lower degree of phospholipid unsaturation in plasma membranes.

Electrophoretic comparison of polypeptides was also made between different membranes. The distribution patterns of polypeptides revealed on one- and two-dimensional SDS-slab gels were characteristic for those membranes. The presence of glycopeptide complements was a useful criterion for distinguishing plasma membrane from other membranes. The plasma membrane and the ER + Golgi membranes were enriched in glycopeptides. However, a marked difference was revealed in the total number and the molecular weights of the peptides.

During cold acclimation of orchard grass seedlings, the degree of fatty acid unsaturation increased only slightly in plasma membrane, unlike in endomembranes. The change in sterols-to-phospholipids ratio in plasma membrane was also slight. On the other hand, the phospholipid-to-protein ratio increased significantly in the plasma membrane as cold hardiness increased. A significant change in the polypeptide complements of plasma membrane was also demonstrated during cold acclimation.

     

Supercooling Ability, Water Content and Hardiness of Rhododendron Flower Buds during Cold Acclimation and Deacclimation

The relationship between supercooling ability and water contentand killing temperature of flower buds during cold acclimationand deacclimation were studied using R. kiusianum and R. x akebono.The occurrence of multiple floret exotherms and their shiftto a narrow range at lower subzero temperatures, as well asthe marked decrease of florets water content, were observedas the symptoms of cold acclimation occuring in flower budsfrom fall to winter, and vice versa in spring buds during deacclimation.In R. kiusianum, the fully acclimated period was from Novemberto March and two months longer than that of R. x akebono. Thesupercooling ability of the former was about –25°Cand about –20°C in the latter. Although the watermigration within bud tissues during the freezing process wasdetermined in the acclimated and deacclimated buds for R. xakebono, no significant water changes could be observed, evenin the acclimated buds. Thus, it is conceivable that deep supercoolingin florets may result not necessarily from water migration fromflorets and bud axes to scales in response to freezing, butfrom low water content in situ of cold-acclimated or artificiallydehydrated flower buds. (Received July 29, 1981; Accepted October 12, 1981)     

Glucolipid Synthesis Activities in Cytoplasmic and Thylakoid Membranes from the Cyanobacterium Anacystis nidulans

Cytoplasmic membranes (plasma membranes), thylakoid membranesand cell walls prepared from the cyanobacterium, Anacystis nidulans,were compared for UDP-glucose: l,2-diacylglycerol glucosyltransferaseactivity. When 1,2-dipalmitoylglycerol was added as a glucosylacceptor, both cytoplasmic membranes and thylakoid membranesincorporated glucose from UDP-glucose into monoglucosyl diacylglycerol,but the cell walls containing the outer membranes did not. Thecytoplasmic membranes incorporated about twice as much glucoseas the thylakoid membranes on a protein basis. These observationssuggest that in A. nidulans the UDP-glucose: 1,2-diacylglycerolglucosyltransferase participating in glucolipid biosynthesisis located in both cytoplasmic and thylakoid membranes, butnot in the outer membrane. ¹Solar Energy Research Group, The Institute of Physical andChemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan. (Received November 21, 1985; Accepted January 27, 1986)     

The Regulation of Proton/Amino Acid Symport in Riccia fluitans L. by Cytosolic pH and Proton Pump Activity

In the aquatic liverwort Riccia fluitans the regulation of theplasma membrane H⁺/amino acid symport has been investigated.Cytosolic pH (pH_c), membrane potential (E_m) and membrane conductancehave been measured and related to transport data, (i) The releaseof [¹⁴C]amino acids is strongly stimulated by cytosolic acidification,induced by the external addition of acetic acid, a decreasein external K⁺, and in the change from light to dark. On average,a decrease in pHc of 0.5 to 0.6 units corresponded with a 4-foldstimulation in amino acid efflux. (ii) External pH changes havefar less effect on substrate transport than the cytosolic pHshifts of the same order. (iii) The inwardly directed positivecurrent, induced by amino acids, is severely inhibited by cytosolicacidification. (iv) Fusicoccin (FC) stimulates amino acid uptakewithout considerable change in proton motive force. (v) Whenthe proton motive force is kept constant, the uptake of aminoacids into Riccia thalli is much lower than when the pump isdeactivated. It is suggested that both the proton pump activityand cytosolic pH are the dominant factors in the regulationof the H⁺/amino acid symport across the plasma membrane of Ricciafluitans, and it is concluded that the proton motive force isnot a reliable quantity to predict and interpret transport kinetics. Key words: Amino acid, cytosolic pH, pH-sensitive electrode, proton motive force, regulation, Riccia fluitans